Compositions comprising female germline stem cells and methods of use thereof

ABSTRACT

The present invention relates to female germline stem cells and their progenitors, methods of isolation thereof, and methods of use thereof.

RELATED APPLICATIONS/PATENTS & INCORPORATION BY REFERENCE

This application claims priority to U.S. Application Ser. No.60/572,222, filed on May 17, 2004 as Attorney Docket No. 910000-3073,U.S. Application Ser. No. 60/574,187, filed on May 24, 2004 as AttorneyDocket No. 910000-3074, and U.S. Application Ser. No. 60/586,641, filedon Jul. 9, 2004 as Attorney Docket No. 910000-3076, the contents each ofwhich are incorporated herein by reference.

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the PCT and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, are hereby expresslyincorporated herein by reference, and may be employed in the practice ofthe invention. More generally, documents or references are cited in thistext, either in a Reference List before the claims, or in the textitself; and, each of these documents or references (“herein citedreferences”), as well as each document or reference cited in each of theherein cited references (including any manufacturer's specifications,instructions, etc.), is hereby expressly incorporated herein byreference.

STATEMENT OF POTENTIAL GOVERNMENT INTEREST

The United States government may have certain rights in this inventionby virtue of grant numbers R01-AG12279 and R01-AG24999 from the NationalInstitute on Aging of the National Institutes of Health.

BACKGROUND OF THE INVENTION

Until recently, it was believed that female gonads of most mammalianspecies, including humans, house a finite number of meiotically-arrestedgerm cells (oocytes) enclosed within primordial follicles that serve asthe stockpile of eggs released at ovulation during each menstrual cycle(Gougeon, A. et al, (1996) Endocr Rev. 17: 121-55; Morita, Y. & Tilly,J. L., (1999) Dev. Biol. 213: 1-17). Oocyte numbers decline throughoutpostnatal life, though mechanisms involving apoptosis (Tilly, J. L.,(2001) Nat. Rev. Mol. Cell Biol. 2: 838-848), which were widely believedto eventually leave the ovaries barren of germ cells (Faddy, M. J. etal., (1976) J. Exp. Zool. 197: 173-186; Faddy, M. J. et al., (1987) CellTissue Kinet. 20: 551-560; Faddy, M. J., (2000) Mol. Cell Endocrinol.163: 43-48). In humans, exhaustion of the oocyte reserve typicallyoccurs during the fifth decade of life, driving menopause. (Richardson,S. J. et al. (1987) J. Clin. Endocrinol. Metab. 65: 1231-1237).

According to this basic doctrine of reproductive biology, it was furtherbelieved that once depleted, the ovarian germ cell pool could not bereplenished. (Zuckerman, S. (1951) Recent Prog. Horm. Res. 6: 63-108;Borum, K., (1961) Exp. Cell Res. 24: 495-507; Peters, H., (1970) Phil.Trans. R. Soc. Lond. B, 259: 91-101; McLaren, A., (1984) Symp. Soc. Exp.Biol. 38: 7-23; Anderson, L. D. and Hirshfield, A. N. (1992) Md. Med. J.41: 614-620). Thus, any treatment that accelerates the loss of oocytesthreatens to decrease the fertility and will cause menopause at anearlier age than expected. For example, exposure of women to a widespectrum of agents that damage the ovary, such as chemotherapeuticagents and radiotherapy, generally leads to premature menopause andirreversible sterility. At present, the limited therapeutic options ofpreserving fertility and normal ovarian function under various adverseconditions are invasive, such as for example cryopreservation of ovariantissue fragments or single oocytes, and often require hormonal therapy,which can be medically inappropriate for many women with hormonallyresponsive tumors (Waxman, J. (1983) J. R. Soc. Med. 76: 144-8;Familiari, G. et al., (1993) Hum. Reprod. 8: 2080-7; Ried, H. L. &Jaffe, N., (1994) Semin. Roentgenol. 29: 6-14; Reichman, B. S. & Green,K. B. (1994) J. Natl. Cancer Inst. Monogr. 16: 125-9). In addition,there are currently no therapeutic options for postponing normal ovarianfailure at menopause. Therefore, there is great need in the art forfurther discovery and development of new or less invasive therapeuticinterventions for restoring failed ovarian function and infertility inwomen.

SUMMARY OF THE INVENTION

It has now been shown that mammalian females do not lose the capacityfor germ-cell renewal during postnatal life. Mammalian ovaries possessmitotically competent female germline stem cells and female germlinestem cell progenitors that, based on rates of oocyte degeneration andclearance, sustain oocyte and follicle production in the postnatalmammalian ovary.

Characterization of female germline stem cells and their progenitorcells are described herein. Accordingly, methods of the invention relateto, among other things, the use of female germline stem cells, and theirprogenitor cells, to expand the follicle reserve as a means of enhancingor restoring fertility in females, and for ameliorating symptoms andconsequences of menopause.

In one aspect, the present invention provides compositions comprisingfemale germline stem cells.

In one embodiment, the present invention provides compositionscomprising female germline stem cells, wherein the cells are mitoticallycompetent and express Vasa, Oct-4, Dazl, Stella and optionally, astage-specific embryonic antigen (“SSEA”). Preferably, the SSEA isSSEA-1. Consistent with their mitotically competent phenotype, femalegermline stem cells of the invention do not expressgrowth/differentiation factor-9 (“GDF-9”), zona pellucida proteins(e.g., zona pellucida protein-3, “ZP3”), histone deacetylase-6 (“HDAC6”)and synaptonemal complex protein-3 (“SCP3”). Upon transplantation into ahost, female germline stem cells of the invention can produce oocytesafter a duration of at least 1 week, more preferably 1 to about 2 weeks,about 2 to about 3 weeks, about 3 to about 4 weeks or more than about 5weeks post transplantation.

In another aspect, the present invention provides compositionscomprising progenitor cells derived from female germline stem cells. Thefemale germline stem cell progenitors (“progenitor cells”) of theinvention are present in the ovary and share common characteristics offemale germline stem cells. Accordingly, in one embodiment, the presentinvention provides compositions comprising female germline stem cellprogenitors, wherein the cells express an SSEA, Vasa, Oct-4, Dazl, andStella, and wherein the cells do not express GDF-9, zona pellucidaproteins (e.g., ZP3), HDAC6 and SCP3. Preferably, the SSEA is SSEA-1.Upon transplantation into a host, female germline stem cell progenitorsof the invention can produce oocytes after a duration of less than 1week, preferably about 24 to about 48 hours post transplantation.

In one embodiment, the present invention provides an isolated cell,wherein the cell is mitotically competent and expresses Vasa, Oct-4,Dazl, Stella and optionally, an SSEA. In a specific embodiment, theisolated cell is a female germline stem cell and in another specificembodiment, the isolated cell is a female germline stem cell progenitorthat expresses SSEA. Preferably, the female germline stem cells, ortheir progenitor cells, are non-embryonic, mammalian, and even morepreferably, human.

In another embodiment, the present invention provides purifiedpopulations of female germline stem cells and/or their progenitor cells.In specific embodiments, the purified population of cells is about 50 toabout 55%, about 55 to about 60%, about 65 to about 70%, about 70 toabout 75%, about 75 to about 80%, about 80 to about 85%, about 85 toabout 90%, about 90 to about 95% or about 95 to about 100% of the cellsin the composition.

In yet another embodiment, the present invention provides pharmaceuticalcompositions comprising female germline stem cells, and/or theirprogenitor cells, and a pharmaceutically acceptable carrier. Thepharmaceutical compositions can comprise purified populations of femalegermline stem cells and/or their progenitor cells.

In another aspect, the present invention provides a method for theisolation of compositions comprising female germline stem cells and/orfemale germline stem cell progenitors, said method comprising the stepsof

-   -   a) homogenizing ovarian tissue;    -   b) contacting the tissue with an agent that binds to an SSEA;        and    -   c) isolating female germline stem cells and/or female germline        stem cell progenitors.

Preferably, the stage-specific embryonic antigen is SSEA-1.

In one embodiment, the present invention provides a method for theisolation of female germline stem cells and/or female germline stem cellprogenitors, said method comprising the steps of

-   -   a) sectioning ovarian tissue;    -   b) labeling the perimeter of the female germline stem cells        and/or female germline stem cell progenitors within the tissue        with an identifying marker;    -   c) applying laser pulses to the perimeter of the female germline        stem cells and/or female germline stem cell progenitors; and    -   d) adhering the female germline stem cells and/or female        germline stem cell progenitors to a capture substrate.

Ovarian tissue can be fresh, frozen or fixed prior to sectioning. Cellscan be labeled with an identifying marker using histological,immunohistochemical, or other compatible techniques to enhance thecontrast between desired and undesired cell types.

In yet another aspect, the invention provides methods for manipulatingfemale germline stem cells, or female germline stem cell progenitors, invivo, ex vivo or in vitro as described herein below.

In one embodiment, the invention provides a method for expanding femalegermline stem cells, or their progenitor cells, in vivo, ex vivo or invitro, comprising contacting female germline stem cells, or theirprogenitor cells, with an agent that increases the amount of femalegermline stem cells, or their progenitor cells, by promotingproliferation or survival thereof, thereby expanding the female germlinestem cells, or their progenitor cells. In a preferred embodiment, theagent includes, but is not limited to, a hormone or growth factor (e.g.,insulin-like growth factor (“IGF”), transforming growth factor (“TGF”),bone morphogenic protein (“BMP”), Wnt protein, or fibroblast growthfactor (“FGF”)), a cell-signaling molecule (e.g.,sphingosine-1-phosphate (“S1P”), or retinoic acid (“RA”)), or apharmacological or pharmaceutical compound (e.g., an inhibitor ofglycogen synthase kinase-3 (“GSK-3”), an inhibitor of apoptosis such asa Bax inhibitor or a caspase inhibitor, an inhibitor of nitric oxideproduction, or an inhibitor of HDAC activity).

In another embodiment, the invention provides a method for identifyingan agent that promotes proliferation or survival of a female germlinestem cell, or its progenitor cell, comprising contacting female germlinestem cells, or their progenitor cells, with a test agent; and detectingan increase in the number of female germline stem cells, or theirprogenitor cells, thereby identifying an agent that promotesproliferation or survival of a female germline stem cell, or itsprogenitor.

In yet another embodiment, the invention provides a method for using thefemale germline stem cells, or their progenitor cells, to characterizepharmacogenetic cellular responses to biologic or pharmacologic agents,comprising isolating female germline stem cells, or their progenitorcells, from a population of subjects, expanding said cells in culture toestablish a plurality of cell cultures, optionally differentiating saidcells into a desired lineage, contacting the cell cultures with one ormore biologic or pharmacologic agents, identifying one or more cellularresponses to the one or more biologic or pharmacologic agents, andcomparing the cellular responses of the cell cultures from differentsubjects.

In yet another embodiment, the invention provides a method for producinga lineage committed cell, comprising contacting a female germline stemcell, or its progenitor cell, with an agent that differentiates thefemale germline stem cell, or its progenitor cell into a lineagecommitted cell. In a preferred embodiment, the agent includes, but isnot limited to, Vascular Endothelial Growth Factor, Sonic Hedgehog,Insulin-like Growth Factor II, Osteogenin, Cytotoxic T CellDifferentiation Factor, b-catenin, Bone Morphogenic Protein 2,Interleukin 2, Transforming Growth Factor b, Nerve Growth Factor,Interleukin 1, Fibroblast Growth Factor 2, Retinoic Acid and Wnt3.

In yet another embodiment, the invention provides a method for reducingthe amount of female germline stem cells, or their progenitor cells, invivo, ex vivo or in vitro, comprising contacting female germline stemcells, or their progenitor cells, with an agent that reduces cellproliferation, thereby reducing the amount of female germline stemcells, or their progenitor cells. In a preferred embodiment, the agentincludes, but is not limited to, a hormone or growth factor (e.g.,TGF-β), a peptide antagonist of mitogenic hormones or growth factors(e.g., the BMP antagonists, Protein Related to DAN and Cerberus (“PRDC”)and Gremlin), or a pharmacological or pharmaceutical compound (e.g., acell cycle inhibitor, or an inhibitor of growth factor signaling).

In yet another embodiment, the invention provides a method for reducingthe amount of female germline stem cells, or their progenitor cells, invivo, ex vivo or in vitro, comprising contacting female germline stemcells, or their progenitor cells, with an agent that inhibits cellsurvival or promotes cell death, thereby reducing the amount of femalegermline stem cells, or their progenitor cells. In a preferredembodiment, the agent the that inhibits cell survival includes, but isnot limited to, a hormone, growth factor or cytokine (e.g., apro-apoptotic tumor necrosis factor (“TNF”) super family member such asTNF-α, Fas-ligand (“FasL”) and TRAIL), an antagonist of pro-survivalBcl-2 family member function, a signaling molecule (e.g., a ceramide),or a pharmacological or pharmaceutical compound (e.g., an inhibitor ofgrowth factor signaling). In a preferred embodiment, the agent the thatpromotes cell death includes, but is not limited to, a pro-apoptotictumor necrosis factor superfamily member (e.g., TNF-α, FasL and TRAIL),agonist of pro-apoptotic Bcl-2 family member function and ceramide.

In yet another embodiment, the invention provides a method foridentifying an agent that reduces proliferation or survival, or promotescell death, of a female germline stem cell, or its progenitor cell,comprising contacting female germline stem cells, or their progenitorcells, with a test agent; and detecting a decrease in the number offemale germline stem cells, or their progenitor cells, therebyidentifying an agent that reduces proliferation or survival, or promotescell death, of a female germline stem cell, or its progenitor cell.

In yet another embodiment, the invention provides a method for oocyteproduction, comprising culturing a female germline stem cell, or itsprogenitor cell, in the presence of an agent that differentiates afemale germline stem cell, or its progenitor cell, into an oocyte,thereby producing an oocyte. In a preferred embodiment, the agentincludes, but is not limited to, a hormone or growth factor (e.g., aTGF, BMP or Wnt family protein, kit-ligand (“SCF”) or leukemiainhibitory factor (“LIF”)), a signaling molecule (e.g.,meiosis-activating sterol, “FF-MAS”), or a pharmacologic orpharmaceutical agent (e.g., a modulator of Id protein function orSnail/Slug transcription factor function).

In yet another embodiment, the invention provides a method for in vitrofertilization of a female subject, said method comprising the steps of:

-   -   a) producing an oocyte by culturing a female germline stem cell,        or its progenitor cell, in the presence of an agent that        differentiates said cell(s) into an oocyte;    -   b) fertilizing the oocyte in vitro to form a zygote; and    -   c) implanting the zygote into the uterus of a female subject.

In yet another embodiment, the invention provides a method for in vitrofertilization of a female subject, said method comprising the steps of:

-   -   a) producing an oocyte by contacting a female germline stem        cell, or its progenitor cell, with an agent that differentiates        said cell(s) into an oocyte;    -   b) fertilizing the oocyte in vitro to form a zygote; and    -   c) implanting the zygote into the uterus of a female subject.

In yet another embodiment, the invention provides a method foridentifying an agent that induces differentiation of a female germlinestem cell, or its progenitor cell, into an oocyte comprising contactingfemale germline stem cells, or their progenitor cells, with a testagent; and detecting an increase in the number of oocytes, therebyidentifying an agent that induces differentiation of a female germlinestem cell, or its progenitor.

In yet another embodiment, the present invention provides a method foroocyte production, comprising providing a female germline stem cell, orits progenitor cell, to a tissue, preferably the ovary, wherein the cellengrafts into the tissue and differentiates into an oocyte, therebyproducing an oocyte.

In yet another embodiment, the present invention provides a method forinducing folliculogenesis, comprising providing a female germline stemcell, or its progenitor cell, to a tissue, preferably the ovary, whereinthe cell engrafts into the tissue and differentiates into an oocytewithin a follicle, thereby inducing folliculogenesis.

In yet another embodiment, the present invention provides a method foroocyte production, comprising contacting ovarian tissue with an agentthat increases the amount of female germline stem cells, or theirprogenitor cells, by promoting proliferation or survival thereof,thereby producing oocytes. In a preferred embodiment, the agentincludes, but is not limited to, a hormone or growth factor (e.g., aIGF, TGF, BMP, Wnt protein or FGF), a cell-signaling molecule (e.g., SPor RA), or a pharmacological or pharmaceutical compound (e.g., aninhibitor of GSK-3, an inhibitor of apoptosis such as a Bax inhibitor orcaspase inhibitor, an inhibitor of nitric oxide production, or aninhibitor of HDAC activity).

In yet another embodiment, the invention provides a method foridentifying an agent that promotes proliferation or survival of a femalegermline stem cell, or its progenitor cell, comprising contactingovarian tissue with a test agent; and detecting an increase in thenumber of female germline stem cells, or their progenitor cells, therebyidentifying an agent that promotes proliferation or survival of a femalegermline stem cell, or its progenitor cell.

In yet another embodiment, the present invention provides a method foroocyte production, comprising contacting ovarian tissue with an agentthat differentiates female germline stem cells, or their progenitorcells, into oocytes, thereby producing oocytes. In a preferredembodiment, the agent can be, but is not limited to, a hormone or growthfactor (e.g., a TGF, BMP, Wnt family protein, SCF or LIF) or apharmacologic or pharmaceutical agent (e.g., a modulator of Id proteinfunction or Snail/Slug transcription factor function).

In yet another embodiment, the invention provides a method foridentifying an agent that induces differentiation of a female germlinestem cell, or its progenitor cell, into an oocyte comprising contactingovarian tissue with a test agent; and detecting an increase in thenumber of oocytes in the ovarian tissue, thereby identifying an agentthat induces differentiation of a female germline stem cell, or itsprogenitor cell.

In yet another embodiment, the present invention provides a method fortreating infertility in a female subject in need thereof comprisingadministering a therapeutically effective amount of a compositioncomprising female germline stem cells, or their progenitor cells, to thesubject, wherein the cells engraft into a tissue, preferably ovariantissue, and differentiate into oocytes, thereby treating infertility.

In yet another embodiment, the present invention provides a method fortreating infertility in a female subject in need thereof comprisingcontacting ovarian tissue of the subject with an agent that increasesthe amount of female germline stem cells, or their progenitor cells, bypromoting proliferation or survival thereof, thereby treatinginfertility in the subject.

In yet another embodiment, the present invention provides a method fortreating infertility in a female subject in need thereof comprisingcontacting ovarian tissue of the subject with an agent thatdifferentiates female germline stem cells, or their progenitor cells,into oocytes, thereby treating infertility in the subject.

In yet another embodiment, the present invention provides a method forrepairing damaged ovarian tissue, comprising providing a therapeuticallyeffective amount of a composition comprising female germline stem cells,or their progenitor cells, to the tissue, wherein the cells engraft intothe tissue and differentiate into oocytes, thereby repairing the damagedtissue. Damage can be caused, for example, by exposure to cytotoxicfactors, chemotherapeutic drugs, radiation, hormone deprivation, growthfactor deprivation, cytokine deprivation, cell receptor antibodies, andthe like. Chemotherapeutic drugs include, but are not limited to,busulfan cyclophosphamide, 5-FU, vinblastine, actinomycin D, etoposide,cisplatin, methotrexate, doxorubicin, among others. Damage can also becaused be diseases that affect ovarian function, including, but notlimited to cancer, polycystic ovary disease, genetic disorders, immunedisorders, metabolic disorders, and the like.

In yet another embodiment, the present invention provides a method forrestoring ovarian function in a menopausal female subject, comprisingadministering a therapeutically effective amount of a compositioncomprising female germline stem cells, or their progenitor cells, to thesubject, wherein the cells engraft into the ovary and differentiate intooocytes, thereby restoring ovarian function. The menopausal femalesubject can be in a stage of either peri- or post-menopause, with saidmenopause caused by either normal (e.g., aging) or pathological (e.g.,surgery, disease, ovarian damage) processes.

In yet another embodiment, the present invention provides a method forrestoring ovarian function in a post-menopausal female subjectcomprising contacting ovarian tissue of the subject with an agent thatincreases the amount of female germline stem cells or their progenitorcells, by promoting proliferation or survival thereof, thereby restoringovarian function in the subject.

In yet another embodiment, the present invention provides a method forrestoring ovarian function in a post-menopausal female subjectcomprising contacting ovarian tissue of the subject with an agent thatdifferentiates female germline stem cells, or their progenitor cells,into oocytes, thereby restoring ovarian function in the subject.

Restoration of ovarian function can relieve adverse symptoms andcomplications associated with menopausal disorders, including, but notlimited to, somatic disorders such as osteoporosis, cardiovasculardisease, somatic sexual dysfunction, hot flashes, vaginal drying, sleepdisorders, depression, irritability, loss of libido, hormone imbalances,and the like, as well as cognitive disorders, such as loss of memory;emotional disorders, depression, and the like.

In yet another embodiment, the present invention provides a method forcontraception in a female subject comprising contacting ovarian tissueof the subject with an agent that decreases the proliferation, functionor survival of female germline stem cells, or their progenitor cells, orthe ability of said cells to produce new oocytes or other somatic celltypes required for fertility, thereby providing contraception to thesubject.

In yet another aspect, the present invention provides kits for use inemploying various agents of the invention.

In one embodiment, the present invention provides a kit for expanding afemale germline stem cell, or its progenitor cell, in vivo, ex vivo orin vitro, comprising an agent that promotes cell proliferation orsurvival of the female germline stem cell, or its progenitor cell, andinstructions for using the agent to promote cell proliferation orsurvival of the female germline stem cell, or its progenitor, therebyexpanding a female germline stem cell, or its progenitor cell inaccordance with the methods of the invention.

In another embodiment, the present invention provides a kit for reducingthe amount of female germline stem cells, or their progenitor cells, invivo, ex vivo or in vitro, comprising an agent that inhibits cellsurvival or promotes cell death and instructions for using the agent toinhibit cell survival or promote cell death of the female germline stemcells, or their progenitor cells, thereby the reducing the amount offemale germline stem cells, or their progenitor cells, in accordancewith the methods of the invention.

In yet another embodiment, the present invention provides a kit foroocyte production, comprising an agent that differentiates a femalegermline stem cell, or its progenitor cell, into an oocyte andinstructions for using the agent to differentiate a female germline stemcell, or its progenitor cell, into an oocyte in accordance with themethods of the invention.

In yet another embodiment, the present invention provides a kit foroocyte production, comprising an agent that increases the amount offemale germline stem cells, or their progenitor cells, by promotingproliferation or survival thereof, and instructions for using the agentto increase the amount of female germline stem cells or their progenitorcells, thereby producing oocytes in accordance with the methods of theinvention.

In yet another embodiment, the present invention provides a kit foroocyte production comprising an agent that differentiates femalegermline stem cells, or their progenitor cells, into oocytes andinstructions for using the agent to differentiate the female germlinestem cells, or their progenitor cells, into oocytes, thereby producingoocytes in accordance with the methods of the invention.

In yet another embodiment, the present invention provides a kit fortreating infertility in a female subject in need thereof comprising anagent that increases the amount of female germline stem cells, or theirprogenitor cells, by promoting proliferation or survival thereof andinstructions for using the agent to increase the amount of femalegermline stem cells or their progenitor cells, thereby treatinginfertility in the subject in accordance with the methods of theinvention.

In yet another embodiment, the present invention provides a kit fortreating infertility in a female subject in need thereof comprising anagent that differentiates female germline stem cells, or theirprogenitor cells, into oocytes, and instructions for using the agent todifferentiate female germline stem cells, or their progenitor cells,into oocytes, thereby treating infertility in the subject in accordancewith the methods of the invention.

In yet another embodiment, the present invention provides a kit forrestoring ovarian function in a post-menopausal female subjectcomprising an agent that increases the amount of female germline stemcells, or their progenitor cells, by promoting proliferation or survivalthereof and instructions for using the agent to increase the amount offemale germline stem cells or their progenitor cells, thereby restoringovarian function in the subject in accordance with the methods of theinvention.

In yet another embodiment, the present invention provides a kit forrestoring ovarian function in a post-menopausal female subjectcomprising an agent that differentiates female germline stem cells, ortheir progenitor cells, into oocytes, and instructions for using theagent to differentiate female germline stem cells, or their progenitorcells, into oocytes, thereby restoring ovarian function in the subjectin accordance with the methods of the invention.

In yet another embodiment, the present invention provides a kit forcontraception in a female subject comprising an agent that decreases theproliferation, function or survival of female germline stem cells, ortheir progenitor cells, or the ability of said cells to produce newoocytes or other somatic cell types required for fertility andinstructions for using the agent to decrease the proliferation, functionor survival of female germline stem cells, or their progenitor cells, orthe ability of said cells to produce new oocytes or other somatic celltypes required for fertility, thereby providing contraception to thesubject in accordance with the methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows graphs depicting postnatal ovarian germ-cell dynamics.Panel (a) shows the numbers of non-atretic follicles, while (b) showsthe numbers of atretic, resting (primordial) and total immature(primordial, primary, small preantral) follicles in mouse ovaries duringpostnatal development. Panel (c) depicts the incidence of primordial andprimary follicle atresia in ovaries exposed to9,10-dimethylbenz[a]anthracene (“DMBA”) on day 25 postpartum. Panel (d)shows the comparison of non-atretic and atretic immature folliclenumbers in C57B1/6, CD1 and AKR/J strains of mice.

FIG. 2 shows the numbers of remaining non-atretic oocytes in rhesusmonkeys.

FIG. 3 shows immunohistochemical and RT-PCR studies of meiotic geneexpression in postnatal mouse ovaries. Panels (a) through (d) depictSPC3 immunostaining in single cells. Panels (e) through (g) show Scp3,Spo11, and Dmc1 expression in ovaries versus testes, or in varioustissues, collected from young adult mice.

FIG. 4 shows postnatal ovarian expression of stem cell-associated genes.The left panel depicts RT-PCR analysis of mili, pumilio-1 (pum1),pumilio-2 (pum2) and nucleostemin expression in mouse ovaries collectedat the indicated days of age or at 8 months (8 m) postpartum. The rightpanel shows tissue distribution analysis of the genes in RNA samplesprepared from ovaries, brains, hearts, kidneys, lungs and spleens offemale mice at 40-42 days of age postpartum.

FIG. 5 depicts busulfan-mediated elimination of the primordial folliclereserve in adult female mice. The graph in (a) shows the numbers ofnon-atretic and atretic primordial follicles present in the ovaries ofvehicle, or busulfan-treated mice. The inset shows results forprimordial follicle atresia. Panels (b) through (e) depict thehistological appearance of ovaries of vehicle-treated orbusulfan-treated mice.

FIG. 6 is a graph showing the ratio of primordial to primary folliclesin evaluation of the long-term outcome of anti-cancer treatment(busulfan) on ovarian function in mice. The calculation of this ratioallows for the estimation of the rate of loss of primordial folliclesvia growth activation.

FIG. 7 depicts generation of new primordial oocytes. FIG. 7 a, b,depicts the number of non-atretic primordial (a) or total immature (b;primordial, primary and small preantral) follicles in ovaries of adultfemale mice at various times following doxorubicin injection (mean±S.E., n=5 mice per treatment group). FIG. 7 c shows graphs depicting theeffect of the broad-spectrum histone deacetylase (HDAC) inhibitor,Trichostatin A (TSA), on the number of non-atretic immature folliclesper ovary of female mice on day 13. The left panel shows the numbers ofnon-atretic immature follicles in response to vehicle or TSA, while theright panel quantifies the numbers of resting (primordial) and earlygrowing (primary and small preantral) follicles. FIG. 7 d shows thenumber of non-atretic primordial, primary and small preantral folliclesin ovaries of female mice on day 241 (d) postpartum, 24 h afterinjection with vehicle or TSA (mean±S. E., n=3-5 mice per treatmentgroup). FIG. 7 e shows that TSA does not reduce the incidence offollicle atresia in mice by showing the number of atretic primordial,primary and small preantral follicles in ovaries of juvenile female mice24 h after injection with either vehicle or TSA (mean±S. E., n=5 miceper treatment group).

FIG. 8 shows that wild-type ovarian tissue adheres to green fluorescentprotein (GFP)-transgenic host ovarian tissue and becomes vascularized.(a, b) Gross morphology of a representative ovarian graft at 3-4 weekspost-surgery, prior to (a) and after (b) removal from the bursal cavity.Panels (c) through (f) show the gross histological appearance ofrepresentative ovarian grafts (broken white line) as viewed under light(c, e) and fluorescence (GFP; d, f) microscopy at 3-4 weekspost-surgery.

FIG. 9 are micrographs showing that GFP-transgenic germ cells formoocytes within follicles in wild-type ovaries. Panels (a) and (b) showGFP expression in sections of host (GFP-transgenic) and grafted(wild-type) ovarian tissues counterstained with propidium iodide. (a)Antral follicle in grafted ovarian tissue containing a GFP-positiveoocyte enclosed within GFP-negative granulosa cells. (b) Primaryfollicle in grafted ovarian tissue containing a GFP-positive oocyteenclosed within GFP-negative granulosa cells (broken white line).

FIG. 10 is an additional example of folliculogenesis in grafted ovariantissue. Two adjacent immature follicles composed of GFP-transgenicoocytes and wild-type granulosa cells present within wild-type (wt)ovarian tissue 4 weeks after grafting into the ovarian bursal cavity ofa GFP-transgenic recipient female (PI, propidium iodidecounterstaining).

FIG. 11 shows graphs depicting oocyte dynamics in Bax deficient (geneknockout, KO) female mice during early postnatal life (Day 4 postpartum)and early reproductive adulthood (Day 42 postpartum).

FIG. 12 shows graphs depicting oocyte dynamics in Caspase-6 deficient(gene knockout, KO) female mice during early postnatal life (Day 4postpartum) and early reproductive adulthood (Day 42 postpartum).

FIG. 13 depicts the representative histology of postpartum day 4wild-type (A, magnified in C) and Atm (ataxia telangiectasia genemutated)-deficient (B, D) ovaries. RT-PCR analysis shows the presence ofgermline markers in both wild-type and Atm-null ovaries (E).

FIG. 14 depicts immunohistochemical analysis of SSEA1 expression (red,with nuclei highlighted by propidium iodide in blue) in adult mouseovaries (B, higher magnification of SSEA1+ cells shown in A; A and C,ovaries from different mice; D single SSEA1+ cell in an adult ovary,showing cell surface expression of the antigen).

FIG. 15 depicts a schematic presentation of one strategy for theisolation of female germline stem cell and/or their progenitors.

FIG. 16 depicts the SSEA-1 isolated (immunopurified) fractionrepresenting a population of cells expressing genes that denotepluripotency (SSEA-1, Oct-4) and places their lineage within thegermline (Dazl, Stella, Mvh/Vasa) but lacking genes expressed in germcells undergoing meiosis (SCP3) or in oocytes (GDF9, ZP3, HDAC6). Theresidual ovarian tissue contains growing oocytes and resting primordialoocytes, and thus all marker genes are expressed in this fraction.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Expansion” refers to the propagation of a cell or cells withoutterminal differentiation. “Isolation phenotype” refers to the structuraland functional characteristics of the female germline stem cells ortheir progenitor cells upon isolation. “Expansion phenotype” refers tothe structural and functional characteristics of the female germlinestem cells or their progenitor cells during expansion. The expansionphenotype can be identical to the isolation phenotype, or alternatively,the expansion phenotype can be more differentiated than the isolationphenotype.

“Differentiation” refers to the developmental process of lineagecommitment. A “lineage” refers to a pathway of cellular development, inwhich precursor or “progenitor” cells undergo progressive physiologicalchanges to become a specified cell type having a characteristic function(e.g., nerve cell, muscle cell or endothelial cell). Differentiationoccurs in stages, whereby cells gradually become more specified untilthey reach full maturity, which is also referred to as “terminaldifferentiation.” A “terminally differentiated cell” is a cell that hascommitted to a specific lineage, and has reached the end stage ofdifferentiation (i.e., a cell that has fully matured). Oocytes are anexample of a terminally differentiated cell type.

The term “isolated” as used herein refers to a female germline stem cellor its progenitor cell, in a non-naturally occurring state (e.g.,isolated from the body or a biological sample from the body). Thebiological sample can include bone marrow, peripheral blood, ovary orspleen.

“Progenitor cells” as used herein are germ lineage cells that are 1)derived from female germline stem cells as the progeny thereof whichcontain a set of common marker genes; 2) are in an early stage ofdifferentiation; and 3) retain mitotic capacity.

“Progeny” as used herein are all daughter cells derived from femalegermline stem cells of the invention, including progenitor cells,differentiated cells, and terminally differentiated cells.

“Derived from” as used herein refers to the process of obtaining adaughter cell.

“Engraft” refers to the process of cellular contact and incorporationinto an existing tissue of interest (e.g., ovary) in vivo.

“Agents” refer to cellular (e.g., biologic) and pharmaceutical factors,preferably growth factors, cytokines, hormones or small molecules, or togenetically-encoded products that modulate cell function (e.g., inducelineage commitment, increase expansion, inhibit or promote cell growthand survival). For example, “expansion agents” are agents that increaseproliferation and/or survival of female germline stem cells or theirprogenitor cells. “Differentiation agents” are agents that induce femalegermline stem cells or their progenitor cells to differentiate intocommitted cell lineages, such as oocytes.

A “follicle” refers to an ovarian structure consisting of a singleoocyte surrounded by somatic (granulosa without or withtheca-interstitial) cells. Somatic cells of the gonad enclose individualoocytes to form follicles. Each fully formed follicle is enveloped in acomplete basement membrane. Although some of these newly formedfollicles start to grow almost immediately, most of them remain in theresting stage until they either degenerate or some signal(s) activate(s)them to enter the growth phase. For reviews on ovarian structure,function and physiology, see Gougeon, A., (1996) Endocr Rev. 17:121-55;Anderson, L. D., and Hirshfield, A. N. (1992) Md Med J. 41: 614-20; andHirshfield, A. N. (1991) Int Rev Cytol. 124: 43-101.

“Mitotically competent” refers to a cell that is capable of mitosis, theprocess by which a cell divides and produces two daughter cells from asingle parent cell.

A “non-embryonic” cell refers to a cell that is obtained from apost-natal source (e.g., infant, child or adult tissue).

A “subject” is a vertebrate, preferably a mammal, more preferably aprimate and most preferably a human. Mammals include, but are notlimited to, primates, humans, farm animals, sport animals, and pets.

The term “obtaining” as in “obtaining the agent” is intended to includepurchasing, synthesizing or otherwise acquiring the agent (or indicatedsubstance or material).

The terms “comprises”, “comprising”, and the like are intended to havethe broad meaning ascribed to them in U.S. Patent Law and can mean“includes”, “including” and the like.

EMBODIMENTS OF THE INVENTION

The present invention provides compositions comprising female germlinestem cells and female germline stem cell progenitors.

Female germline stem cells express markers including Vasa, Oct-4, Dazl,Stella and optionally an SSEA. Female germline stem cells aremitotically competent (i.e., capable of mitosis) and accordingly, do notexpress growth/differentiation factor-9 (“GDF-9”), zona pellucidaproteins (e.g., zona pellucida protein-3, “ZP3”), histone deacetylase-6(“HDAC6”) or synaptonemal complex protein-3 (“SCP3”). For additionaldetails, see, U.S. application Ser. Nos. ______, filed on May 17, 2005as Attorney Docket No. 51588-62060, and ______, filed on May 17, 2005 asAttorney Docket No. 51588-62065, the contents of which are incorporatedherein by reference for their description of female germline stem cellsin the bone marrow and peripheral blood.

The present invention also provides progenitor cells derived from femalegermline stem cells. Female germline stem cell progenitors of theinvention can circulate throughout the body and most preferably can belocalized in bone marrow, peripheral blood and ovary. Progenitor cellsof the invention express an SSEA, Oct-4, Vasa, Dazl and Stella but donot express HDAC6, GDF-9, and zona pellucida proteins (e.g., ZP3) orSCP3. Preferably, the SSEA is SSEA-1.

Female germline stem cells and female germline stem cell progenitors ofthe invention have functional distinctions. Upon transplantation into ahost, female germline stem cells of the invention can produce oocytesafter a duration of at least 1 week, more preferably 1 to about 2 weeks,about 2 to about 3 weeks, about 3 to about 4 weeks or more than about 5weeks post transplantation. Female germline stem cell progenitors havethe capacity to generate oocytes more rapidly than female germline stemcells. Upon transplantation into a host, female germline stem cellprogenitors of the invention can produce oocytes after a duration ofless than 1 week, preferably about 24 to about 48 hours posttransplantation. For additional details, see, U.S. application Ser. No.______, filed on May 17, 2005 as Attorney Docket No. 51588-62060, thecontents of which are incorporated herein by reference for theirdescription of post transplantation oocyte production.

Oct-4 is a gene expressed in female germline stem cells and theirprogenitor cells. The Oct-4 gene encodes a transcription factor that isinvolved in the establishment of the mammalian germline and plays asignificant role in early germ cell specification (reviewed in Scholer(1991), Trends Genet. 7(10): 323-329). In the developing mammalianembryo, Oct-4 is down-regulated during the differentiation of theepiblast, eventually becoming confined to the germ cell lineage. In thegermline, Oct-4 expression is regulated separately from epiblastexpression. Expression of Oct-4 is a phenotypic marker of totipotency(Yeom et al. (1996); Development 122: 881-888).

Stella is a gene expressed in female germline stem cells and theirprogenitor cells. Stella is a novel gene specifically expressed inprimordial germ cells and their descendants, including oocytes (Bortvinet al. (2004) BMC Developmental Biology 4(2):1-5). Stella encodes aprotein with a SAP-like domain and a splicing factor motif-likestructure. Embryos deficient in Stella expression are compromised inpreimplantation development and rarely reach the blastocyst stage. Thus,Stella is a maternal factor implicated in early embryogenesis.

Dazl is a gene expressed in female germline stem cells and theirprogenitor cells. The autosomal gene Dazl is a member of a family ofgenes that contain a consensus RNA binding domain and are expressed ingerm cells. Loss of expression of an intact Dazl protein in mice isassociated with failure of germ cells to complete meiotic prophase.Specifically, in female mice null for Dazl, loss of germ cells occursduring fetal life at a time coincident with progression of germ cellsthrough meiotic prophase. In male mice null for Dazl, germ cells wereunable to progress beyond the leptotene stage of meiotic prophase I.Thus, in the absence of Dazl, progression through meiotic prophase isinterrupted (Saunders et al. (2003), Reproduction, 126:589-597).

Vasa is a gene expressed in female germline stem cells and theirprogenitor cells. Vasa is a component of the germplasm that encodes aDEAD-family ATP-dependent RNA helicase (Liang et al. (1994) Development,120:1201-1211; Lasko et al. (1988) Nature, 335:611-167). The molecularfunction of Vasa is directed to binding target mRNAs involved in germcell establishment (e.g., Oskar and Nanos), oogenesis, (e.g., Gruken),and translation onset (Gavis et al. (1996) Development, 110: 521-528).Vasa is required for pole cell formation and is exclusively restrictedto the germ cell lineage throughout the development. Thus, Vasa is amolecular marker for the germ cell lineage in most animal species(Toshiaki et al. (2001) Cell Structure and Function 26:131-136).

Stage-Specific Embryonic Antigens are optionally expressed in femalegermline stem cells and expressed in female germline stem cellprogenitors of the invention. Stage-Specific Embryonic Antigen-1(SSEA-1) is a cell surface embryonic antigen whose functions areassociated with cell adhesion, migration and differentiation. Duringhypoblast formation, SSEA-1 positive cells can be identified in theblastocoel and hypoblast and later in the germinal crescent. SSEA-1functions in the early germ cell and neural cell development. (D'Costaet al. (1999) Int J. Dev. Biol. 43(4): 349-356; Henderson et al. (2002)Stem Cells 20: 329-337). In specific embodiments, expression of SSEAs infemale germline stem cells may arise as the cells differentiate.

Female germline stem cells and their progenitor cells do not expressGDF-9, a gene expressed in cells that have already started todifferentiate into oocytes. Growth/differentiation factor-9 (GDF-9) is amember of the transforming growth factor-β superfamily, highly expressedin ovaries. GDF-9 mRNA can be found in neonatal and adult oocytes fromthe primary one-layer follicle stage until after ovulation (Dong, J. etal (1996) Nature 383: 531-5). Analysis of GDF-9 deficient mice revealsthat only primordial and primary one-layer follicles can be formed, buta block beyond the primary one-layer follicle stage in folliculardevelopment occurs, resulting in complete infertility.

Female germline stem cells and their progenitor cells do not expressZP1, ZP2, and ZP3, which are gene products that comprise the zonapellucida (ZP) of the oocyte. Their expression is regulated by a basichelix-loop-helix (bHLH) transcription factor, FIGα. Mice null in FIGα donot express the Zp genes and do not form primordial follicles (Soyal, S.M., et al (2000) Development 127: 4645-4654). Individual knockouts ofthe ZP genes result in abnormal or absent zonae pellucidae and decreasedfertility (ZP1; Rankin T, et al (1999) Development. 126: 3847-55) orsterility (ZP2, Rankin T L, et al. (2001) Development 128: 1119-26; ZP3,Rankin T et al (1996) Development 122: 2903-10). The ZP protein productsare glycosylated, and subsequently secreted to form an extracellularmatrix, which is important for in vivo fertilization andpre-implantation development. Expression of the ZP proteins is preciselyregulated and restricted to a two-week growth phase of oogenesis. ZPmRNA transcripts are not expressed in resting oocytes, however once theoocytes begin to grow, all three ZP transcripts begin to accumulate.

Female germline stem cells and their progenitor cells do not expressHDAC6. HDACs, or histone deacetylases are involved in ovarian follicledevelopment. HDAC6 in particular can be detected in resting germinalvesicle-stage (primordial) oocytes (Verdel, A., et al. (2003) Zygote 11:323-8; FIG. 16). HDAC6 is a class II histone deacetylase and has beenimplicated as a microtubule-associated deactylase (Hubbert, C. et al,(2002) Nature 417: 455-8). HDACs are the target of inhibitors including,but not limited to, trichostatin A and trapoxin, both of which aremicrobial metabolites that induce cell differentiation, cell cyclearrest, and reversal of the transformed cell morphology.

Female germline stem cells and their progenitor cells do not expressSCP3, consistent with observations that they are pre-meiotic stem cells(i.e., diploid). The synaptonemal complex protein SCP3 is part of thelateral element of the synaptonemal complex, a meiosis-specific proteinstructure essential for synapsis of homologous chromosomes. Thesynaptonemal complex promotes pairing and segregation of homologouschromosomes, influences the number and relative distribution ofcrossovers, and converts crossovers into chiasmata. SCP3 ismeiosis-specific and can form multi-stranded, cross-striated fibers,forming an ordered, fibrous core in the lateral element (Yuan, L. et al,(1998) J. Cell. Biol. 142: 331-339). The absence of SCP3 in mice canlead to female germ cell aneuploidy and embryo death, possibly due to adefect in structural integrity of meiotic chromosomes (Yuan, L. et al,(2002) Science 296: 1115-8). Female germline stem cells and theirprogenitor cells can be isolated from ovarian homogenate usingimmuno-affinity separation with the Stage-Specific Embryonic Antigen-1antibody (“anti-SSEA-1”) (commercially available, for example, fromChemicon (MAB4301)).

Methods of antibody based separation and isolation generally known inthe art can be employed to obtain SSEA-1 positive germ cells fromovarian homogenate. In one embodiment, magnetic beads can be used in theseparation procedure. For example, the CELLection biotin binder kit andmagnetic device from Dynal Biotech can be used to isolate the SSEA-1positive cells. Biotinylated anti-SSEA-1 antibodies can be attached tocoated magnetic beads and combined with cellular homogenate, thecombination of which is subsequently fractionated by magneticseparation. Post-isolation, the affinity beads can be removed. Aliquotsof isolated cells can additionally be collected and separated by flowcytometry. Multi-step cell isolation techniques can maximize thepreparation of live cells for subsequent culture and manipulation,freezing, and/or transplantation.

Germline stem cell and their progenitors can also be isolated fromovarian homogenate using laser-capture microdissection. Using thistechnique, female germline stem cells are obtained from sectionedovarian tissue. Ovarian tissue can be fresh, frozen or fixed prior tosectioning. Laser-capture microdissection is then carried out to isolatethe female germline stem cells. The procedure of laser capturemicrodissection is well known in the art, see, for example, Eltoum I Aet al., (2002) Adv. Anat. Pathol. 9: 316-322).

Laser capture microdissection makes use of a laser pulsing apparatus inconjunction with a specially-adapted microscope and real-timevisualization computer system. First, target cells or cell types withina heterogeneous tissue section on a histological slide are identifiedand “marked” by labeling their perimeter via a computer interface. Thesecells may have been specifically labeled using histological,immunohistochemical, or other compatible techniques to enhance thecontrast between desired and undesired cell types. Laser pulses are thenapplied to the perimeter of the cells to be captured as specified. Laserpulsing most often results in the adherence of desired, “marked” cellsto a proprietary capture substrate, while undesired cells are excludedand remain attached to the histological slide. Cells attached to thecapture substrate are then processed for downstream analyses, (e.g.,analysis of gene expression in specific cell types within a tissue).

Female germline stem cells and their progenitor cells can be isolated bystandard means known in the art for the separation of stem cells fromthe blood and marrow (e.g., cell sorting). Preferably, the isolationprotocol includes generation of a kit⁺/lin⁻ fraction that is depleted ofhematopoietic cells. Additional selection means based on the uniqueprofile of gene expression (e.g., Vasa, Oct-4, Dazl, and Stella) can beemployed to further purify populations of cells comprising femalegermline stem cells and their progenitor cells. Compositions comprisingfemale germline stem cells and their progenitor cells can be isolatedand subsequently purified to an extent where they become substantiallyfree of the biological sample from which they were obtained (e.g. bonemarrow, peripheral blood, ovary).

Female germline stem cell progenitors can be obtained from femalegermline stem cells by, for example, expansion in culture. Thus, theprogenitor cells can be cells having an “expansion phenotype.”

I. Administration

The present invention provides compositions comprising female germlinestem cells, or progenitor cells derived from female germline stem cells.The compositions can be pharmaceutical compositions comprising femalegermline stem cells, or progenitor cells derived from female germlinestem cells and a pharmaceutically acceptable carrier.

Compositions of female germline stem cells, or progenitors derived fromfemale germline stem cells, can be provided directly to a tissue, suchas ovarian tissue. Following transplantation or implantation, the cellscan engraft and differentiate into oocytes. “Engraft” refers to theprocess of cellular contact and incorporation into an existing tissue ofinterest (e.g., ovary) in vivo. Expansion and differentiation agents canbe provided prior to, during or after administration to increase theamount of oocytes in vivo.

Administration can be autologous or heterologous allogenic). Forexample, female germline stem cells, or progenitors derived from femalegermline stem cells, can be obtained from one subject, and administeredto the same or a different subject.

Preferably, the engrafted cells undergo oogenesis followed byfolliculogenesis, wherein the cells differentiate into an oocyte withina follicle. Folliculogenesis is a process in which an ovarian structureconsisting of a single oocyte is surrounded by somatic (granulosawithout or with theca-interstitial) cells. Somatic cells of the gonadenclose individual oocytes to form follicles. Each fully formed follicleis enveloped in a complete basement membrane. Although some of thesenewly formed follicles start to grow almost immediately, most of themremain in the resting stage until they either degenerate or somesignal(s) activate(s) them to enter the growth phase.

Germline stem cells of the invention or their progeny (e.g.,progenitors, differentiated progeny and terminally differentiatedprogeny) can be administered via localized injection, including catheteradministration, systemic injection, localized injection, intravenousinjection, intrauterine injection or parenteral administration. Whenadministering a therapeutic composition of the present invention (e.g.,a pharmaceutical composition), it will generally be formulated in a unitdosage injectable form (solution, suspension, emulsion).

Compositions of the invention can be conveniently provided as sterileliquid preparations, e.g., isotonic aqueous solutions, suspensions,emulsions, dispersions, or viscous compositions, which may be bufferedto a selected pH. Liquid preparations are normally easier to preparethan gels, other viscous compositions, and solid compositions.Additionally, liquid compositions are somewhat more convenient toadminister, especially by injection. Viscous compositions, on the otherhand, can be formulated within the appropriate viscosity range toprovide longer contact periods with specific tissues. Liquid or viscouscompositions can comprise carriers, which can be a solvent or dispersingmedium containing, for example, water, saline, phosphate bufferedsaline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cellsutilized in practicing the present invention in the required amount ofthe appropriate solvent with various amounts of the other ingredients,as desired. Such compositions may be in admixture with a suitablecarrier, diluent, or excipient such as sterile water, physiologicalsaline, glucose, dextrose, or the like. The compositions can also belyophilized. The compositions can contain auxiliary substances such aswetting, dispersing, or emulsifying agents (e.g., methylcellulose), pHbuffering agents, gelling or viscosity enhancing additives,preservatives, flavoring agents, colors, and the like, depending uponthe route of administration and the preparation desired. Standard texts,such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985,incorporated herein by reference, may be consulted to prepare suitablepreparations, without undue experimentation.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example;aluminum monostearate and gelatin. According to the present invention,however, any vehicle, diluent, or additive used would have to becompatible with the germline stem cells or their progenitors.

The compositions can be isotonic, i.e., they can have the same osmoticpressure as blood and lacrimal fluid. The desired isotonicity of thecompositions of this invention may be accomplished using sodiumchloride, or other pharmaceutically acceptable agents such as dextrose,boric acid, sodium tartrate, propylene glycol or other inorganic ororganic solutes. Sodium chloride is preferred particularly for bufferscontaining sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the agent selected. Theimportant point is to use an amount that will achieve the selectedviscosity. Obviously, the choice of suitable carriers and otheradditives will depend on the exact route of administration and thenature of the particular dosage form, e.g., liquid dosage form (e.g.,whether the composition is to be formulated into a solution, asuspension, gel or another liquid form, such as a time release form orliquid-filled form).

A method to potentially increase cell survival when introducing thecells into a subject in need thereof is to incorporate germline stemcells or their progeny (e.g., in vivo, ex vivo or in vitro derived) ofinterest into a biopolymer or synthetic polymer. Depending on thesubject's condition, the site of injection might prove inhospitable forcell seeding and growth because of scarring or other impediments.Examples of biopolymer include, but are not limited to, cells mixed withfibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans.This could be constructed with or without included expansion ordifferentiation factors. Additionally, these could be in suspension, butresidence time at sites subjected to flow would be nominal. Anotheralternative is a three-dimensional gel with cells entrapped within theinterstices of the cell biopolymer admixture. Again, expansion ordifferentiation factors could be included with the cells. These could bedeployed by injection via various routes described herein.

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert and will notaffect the viability or efficacy of the germline stem cells or theirprogenitors as described in the present invention. This will present noproblem to those skilled in chemical and pharmaceutical principles, orproblems can be readily avoided by reference to standard texts or bysimple experiments (not involving undue experimentation), from thisdisclosure and the documents cited herein.

One consideration concerning the therapeutic use of germline stem cellsand their progeny is the quantity of cells necessary to achieve anoptimal effect. In current human studies of autologous mononuclear bonemarrow cells, empirical doses ranging from 1 to 4×10⁷ cells have beenused with encouraging results. However, different scenarios may requireoptimization of the amount of cells injected into a tissue of interest,such as ovarian tissue. Thus, the quantity of cells to be administeredwill vary for the subject being treated. Preferably, between 10² to 10⁶,more preferably 10³ to 10⁵, and still more preferably, 10⁴ stem cellscan be administered to a human subject. However, the precisedetermination of what would be considered an effective dose may be basedon factors individual to each patient, including their size, age, sex,weight, and condition of the particular patient. As few as 100-1000cells can be administered for certain desired applications amongselected patients. Therefore, dosages can be readily ascertained bythose skilled in the art from this disclosure and the knowledge in theart.

Another consideration regarding the use of germline stem cells or theirprogenitors is the purity of the population. Ovarian cells, for example,comprise mixed populations of cells, which can be purified to a degreesufficient to produce a desired effect. Those skilled in the art canreadily determine the percentage of germline stem cells or theirprogenitors in a population using various well-known methods, such asfluorescence activated cell sorting (FACS). Preferable ranges of purityin populations comprising germline stem cells or their progenitors areabout 50 to about 55%, about 55 to about 60%, and about 65 to about 70%.More preferably the purity is about 70 to about 75%, about 75 to about80%, about 80 to about 85%; and still more preferably the purity isabout 85 to about 90%, about 90 to about 95%, and about 95 to about100%. Purity of the germline stem cells or their progenitors can bedetermined according to the genetic marker marker profile within apopulation. Dosages can be readily adjusted by those skilled in the art(e.g., a decrease in purity may require an increase in dosage).

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions and to beadministered in methods of the invention. Typically, any additives (inaddition to the active stem cell(s) and/or agent(s)) are present in anamount of 0.001 to 50% (weight) solution in phosphate buffered saline,and the active ingredient is present in the order of micrograms tomilligrams, such as about 0.0001 to about 5 wt %, preferably about0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05 wt %or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %,and still more preferably about 0.05 to about 5 wt %. Of course, for anycomposition to be administered to an animal or human, and for anyparticular method of administration, it is preferred to determinetherefore: toxicity, such as by determining the lethal dose (LD) andLD₅₀ in a suitable animal model e.g., rodent such as mouse; and, thedosage of the composition(s), concentration of components therein andtiming of administering the composition(s), which elicit a suitableresponse. Such determinations do not require undue experimentation fromthe knowledge of the skilled artisan, this disclosure and the documentscited herein. And, the time for sequential administrations can beascertained without undue experimentation.

II. Germline Stem Cell Modulation and Oocyte Production

The present invention provides methods for oocyte production, in vivo,in vitro and ex-vivo. Oocyte production can be increased by increasingthe number of female germline stem cells, or progenitors derived fromfemale germline stem cells. The number of female germline stem cells, orprogenitors derived from female germline stem cells can be increased byincreasing the survival or proliferation of existing female germlinestem cells, or progenitors derived from female germline stem cells.

Agents (e.g., expansion agents) which increase proliferation or survivalof female germline stem cells, or progenitors derived from femalegermline stem cells include, but are not limited to, a hormone or growthfactor (e.g., a IGF, TGF, BMP, Wnt protein or FGF), a cell-signalingmolecule (e.g., SIP or RA), or a pharmacological or pharmaceuticalcompound (e.g., an inhibitor of GSK-3, an inhibitor of apoptosis such asa Bax inhibitor or caspase inhibitor, an inhibitor of nitric oxideproduction, or an inhibitor of HDAC activity).

Agents comprising growth factors are known in the art to increaseproliferation or survival of stem cells. For example, U.S. Pat. Nos.5,750,376 and 5,851,832 describe methods for the in vitro culture andproliferation of neural stem cells using TGF. An active role in theexpansion and proliferaion of stem cells has also been described forBMPs (Zhu, G. et al, (1999) Dev. Biol. 215: 118-29 and Kawase, E. et al,(2001) Development 131: 1365) and Wnt proteins (Pazianos, G. et al,(2003) Biotechniques 35: 1240 and Constantinescu, S. (2003) J. Cell Mol.Med. 7: 103). U.S. Pat. Nos. 5,453,357 and 5,851,832 describeproliferative stem cell culture systems that utilize FGFs. The contentsof each of these references are specifically incorporated herein byreference for their description of expansion agents known in the art.

Agents comprising cell-signaling molecules are also known in the art toincrease proliferation or survival of stem cells. For example,Sphingosine-1-phosphate is known to induce proliferation of neuralprogenitor cells (Harada, J. et al, (2004) J. Neurochem. 88: 1026). U.S.Patent Application No. 20030113913 describes the use of retinoic acid instem cell self renewal in culture. The contents of each of thesereferences are specifically incorporated herein by reference for theirdescription of expansion agents known in the art.

Agents comprising pharmacological or pharmaceutical compounds are alsoknown in the art to increase proliferation or survival of stem cells.For example, inhibitors of glycogen synthase kinase maintainpluripotency of embryonic stem cells through activation of Wnt signaling(Sato, N. et al, (2004) Nat. Med. 10: 55-63). Inhibitors of apoptosis(Wang, Y. et al, (2004) Mol. Cell. Endocrinol. 218: 165), inhibitors ofnitric oxide/nitric oxide synthase (Matarredona, E. R. et al, (2004)Brain Res. 995: 274) and inhibitors of histone deacetylases (Lee, J. H.et al, (2004) Genesis 38: 32-8) are also known to increase proliferationand/or pluripotency. For example, the peptide humanin is an inhibitor ofBax function that suppresses apoptosis (Guo, B. et al, (2003) Nature423: 456-461). The contents of each of these references are specificallyincorporated herein by reference for their description of expansionagents known in the art.

Oocyte production can be further increased by contacting compositionscomprising female germline stem cells, or progenitors derived fromfemale germline stem cells, with an agent that differentiates femalegermline stem cells or their progenitors into oocytes (e.g.,differentiation agents). Such differentiation agents include, but arenot limited to, a hormone or growth factor (e.g., TGF, BMP, Writprotein, SCF or LIF), a signaling molecule (e.g., meiosis-activatingsterol, “FF-MAS”), or a pharmacologic or pharmaceutical agent (e.g., amodulator of Id protein function or Snail/Slug transcription factorfunction).

Agents comprising growth factors are known in the art to inducedifferentiation of stem cells. For example, TGF-β can inducedifferentiation of hematopoietic stem cells (Ruscetti, F. W. et al,(2001) Int. J. Hematol. 74: 18-25). U.S. Patent Application No.2002142457 describes methods for differentiation of cardiomyocytes usingBMPs. Pera et al describe human embryonic stem cell differentiationusing BMP-2 (Pera, M. F. et al, (2004) J. Cell Sci. 117: 1269). U.S.Patent Application No. 20040014210 and U.S. Pat. No. 6,485,972 describemethods of using Wnt proteins to induce differentiation. U.S. Pat. No.6,586,243 describes differentiation of dendritic cells in the presenceof SCF. U.S. Pat. No. 6,395,546 describes methods for generatingdopaminergic neurons in vitro from embryonic and adult central nervoussystem cells using LIF. The contents of each of these references arespecifically incorporated herein by reference for their description ofdifferentiation agents known in the art.

Agents comprising signaling molecules are also known to inducedifferentiation of oocytes. FF-Mas is known to promote oocyte maturation(Marin Bivens, C. L. et al, (2004) BOR papers in press). The contents ofeach of these references are specifically incorporated herein byreference for their description of differentiation agents known in theart.

Agents comprising pharmacological or pharmaceutical compounds are alsoknown in the art to induce differentiation of stem cells. For example,modulators of Id are involved in hematopoietic differentiation(Nogueria, M. M. et al, (2000) 276: 803) and Modulators of Snail/Slugare known to induce stem cell differentiation (Le Douarin, N. M. et al,(1994) Curr. Opin. Genet. Dev. 4: 685-695; Plescia, C. et al, (2001)Differentiation 68: 254-69). The contents of each of these referencesare specifically incorporated herein by reference for their descriptionof differentiation agents known in the art.

The present invention also provides methods for reducing female germlinestem cells, or progenitors derived from female germline stem cells, invivo, ex vivo or in vitro, comprising contacting female germline stemcells or their progenitor cells with an agent that reduces cellproliferation, inhibits cell survival or promotes cell death. Unwantedproliferation of the cells of the invention can give rise to cancerousand pre-cancerous phenotypes (e.g., germ cell tumors, ovarian cancer).Such methods can be used to control unwanted proliferation (e.g.,cancer) or for contraceptive measures by reducing the numbers ofgermline stem cells, and optionally their progenitors or oocytes.

Agents that reduce cell proliferation include, but are not limited to, ahormone or growth factor (e.g., TGF-β), a peptide antagonist ofmitogenic hormones or growth factors (e.g., the BMP antagonists, PRDCand Gremlin), or a pharmacological or pharmaceutical compound (e.g., acell cycle inhibitor, or an inhibitor of growth factor signaling).

Agents that inhibit cell survival include, but are not limited to, ahormone, growth factor or cytokine (e.g., a pro-apoptotic TNF superfamily member such as TNF-α, FasL and TRAIL), an antagonist ofpro-survival Bcl-2 family member function, a signaling molecule (e.g., aceramide), or a pharmacological or pharmaceutical compound (e.g., aninhibitor of growth factor signaling). Pro-survival Bcl-2 family membersinclude Bcl-2, Bcl-xl (Cory, S. and Adams, J. M. (2000) Nat Rev Cancer2(9):647-656; Lutz, R. J. (2000) Cell Survival Apoptosis 28:51-56),Bcl-W (Gibson, L., et al. (1996) Oncogene 13, 665-675; Cory, S. andAdams, J. M. (2000) Nat Rev Cancer 2(9):647-656), Mcl-1 (Kozopas, K. M.,et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:3516-3520; Reynolds, J.E., et al. (1994) Cancer Res. 54:6348-6352; Cory, S. and Adams, J. M.(2000) Nat Rev Cancer 2(9):647-656) and A1 (Cory, S. and Adams, J. M.(2000) Nat Rev Cancer 2(9):647-656; Gonzales, J., et al. (2003) Blood101(7):2679-2685; Reed, J. C. (1997) Nature 387:773-776).

Agents that promote cell death include, but are not limited to, apro-apoptotic tumor necrosis factor superfamily member (e.g., TNF-α,FasL and TRAIL), agonist of pro-apoptotic Bcl-2 family member functionand ceramide. Pro-apoptotic Bcl-2 family members include Bax (Oltvai, ZN, et al. (1993): Cell 74: 609-619), Bak (Chittenden, T, et al. (1995)Nature 374:733-736), Bid (Luo, X., et al. (1998) Cell 94:481-490), Hrk(Inohara, N. et al. (1997) EMBO J 16(7):1686-1694), Bod (Hsu, et al.(1998) Mol Endocrinol. 12(9):1432-1440), Bim (O'Connor, L., et al.(1998) EMBO J. 17(2):385-395), Noxa (Oda, E., et al. (2000) Science 288,1053-1058; Yakovlev, A. G., et al. (2004) J Biol Chem279(27):28367-28374), puma (Nakano, K. and Vousden, K. H. (2001) MolCell 7(3):683-694), Bok (Yakovlev, A. G., et al. (2004) J Biol Chem279(27):28367-28374; Hsu, S Y, et al. (1997) Proc Natl Acad Sci USA.94(23):12401-6) and Bcl-xs (Boise, L. H., et al. (1993) Cell74:597-608).

Several agents are known in the art to inhibit cell proliferation orsurvival or promote cell death, including PRDC (Sudo et al, (2004) J.Biol. Chem., advanced publication), TNF (Wong, G. et al, (2004) Exp.Neurol. 187: 171), FasL (Sakata, S. et al, (2003) Cell Death Differ. 10:676) and TRAIL (Pitti, R M, et al. (1996) J Biol Chem 271: 12687-12690;Wiley, S R, et al. (1995) Immunity 3: 673-682). Ceramide mediates theaction of tumor necrosis factor on primitive human hematopoietic cells(Maguer-Satta, V. et al, (2000) Blood 96: 4118-23). Agonist/antagonistof Bcl-2 family members, such as Bcl-2, Bcl-XL, Bcl-W, Mcl-1, A1, Bax,Bak, Bid, Hrk, Bod, Bim, Noxa, Puma, Bok and Bcl-xs, are known toinhibit stem cell survival (Lindsten, T. et al, (2003) J. Neurosci. 23:11112-9). Agents comprising pharmacological or pharmaceutical compoundsare also known in the art to inhibit cell survival. For example,inhibitors of growth factor signaling, such as QSulf1, a heparan sulfate6-O-endosulfatase that inhibits fibroblast growth factor signaling, caninhibit stem cell survival (Wang, S. et al, (2004) Proc. Natl. Acad.Sci. USA 101: 4833). The contents of each of these references arespecifically incorporated herein by reference for their description ofagents known in the art to inhibit cell survival.

Agents can be administered to subjects in need thereof by a variety ofadministration routes. Methods of administration, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,intraocular, buccal, intravaginal, intracisternal,intacerebroventricular, intratracheal, nasal, transdermal, within/onimplants, e.g., fibers such as collagen, osmotic pumps, or graftscomprising appropriately transformed cells, etc., or parenteral routes.The term “parenteral” includes subcutaneous, intravenous, intramuscular,intraperitoneal, or infusion. Intravenous or intramuscular routes arenot particularly suitable for long-term therapy and prophylaxis. Aparticular method of administration involves coating, embedding orderivatizing fibers, such as collagen fibers, protein polymers, etc.with therapeutic proteins. Other useful approaches are described inOtto, D. et al., J. Neurosci. Res. 22: 83-91 and in Otto, D. andUnsicker, K. J. Neurosci. 10: 1912-1921.

In vitro and ex vivo applications can involve culture of the germlinestem cells or their progenitors with the selected agent to achieve thedesired result.

Agents of the invention may be supplied along with additional reagentsin a kit. The kits can include instructions for the treatment regime orassay, reagents, equipment (test tubes, reaction vessels, needles,syringes, etc.) and standards for calibrating or conducting thetreatment or assay. The instructions provided in a kit according to theinvention may be directed to suitable operational parameters in the formof a label or a separate insert. Optionally, the kit may furthercomprise a standard or control information so that the test sample canbe compared with the control information standard to determine ifwhether a consistent result is achieved.

III. Culture

Germline stem cells of the invention, and progenitors derived fromgermline stem cells, can be used for many diverse clinical andpre-clinical applications, which can include, but are not limited to,experimental use in toxicological or genomic screening methods, as wellas treatment of the diseases disclosed herein.

The present invention provides methods for expanding female germlinestem cells, or progenitors derived from female germline stem cells, invitro, comprising contacting a female germline stem cell, or itsprogenitor, with an agent that promotes cell proliferation or survival.Expansion agents can be the same as are used in vivo and ex vivo, andinclude, but are not limited to, a hormone or growth factor (e.g., aIGF, TGF, BMP, Wnt protein or FGF), a cell-signaling molecule (e.g., S1Por RA), or a pharmacological or pharmaceutical compound (e.g., aninhibitor of GSK-3, an inhibitor of apoptosis such as a Bax inhibitor orcaspase inhibitor, an inhibitor of nitric oxide production, or aninhibitor of HDAC activity).

Female germline stem cells and their progenitors can providedifferentiated and imdifferentiated cultured cell types forhigh-throughput toxicological or genomic screening as well astherapeutic use. The cells can be cultured in, for example, 96-well orother multi-well culture plates to provide a system for scale-up andhigh-throughput screening of, for example, target cytokines, chemokines,growth factors, or pharmaceutical compositions in pharmacogenomics orpharmacogenetics. Cytokines, hormones, pharmaceutical compositions andgrowth factors, for example, can therefore be screened in a timely andcost-effective manner to more clearly elucidate their effects.

Germline stem cells of the invention, or progenitors derived fromgermline stem cells, further provide a unique system in which cells canbe differentiated to form specific cell lineages (e.g., oocytes).Cultures of cells (from the same individual and from differentindividuals) can be treated with differentiation agents of interest tostimulate the production of oocytes, which can then be used for avariety of therapeutic applications (e.g., in vitro fertilization,somatic cell nuclear transfer).

Modulation of the properties of female germline stem cell or theirprogenitors, such as their proliferation rate, their rate of death,their differentiation into oocytes or other cell types, their longevity,their suitability for handling, transplantation, culture, preservation,or other properties, can be assessed in culture. Isolated cells can becultured in a range of media suitable for cell culture. Additivesinclude but are not be limited to serum, antibiotics (if needed), andbioactive molecules like LIF, Kit ligand, and βFGF, Flt-3 ligand, etc.

Differentiation of female germline stem cells or their progenitors, asrepresented by meiotic entrance and oocyte development, or developmentinto other cell lineages, including somatic cells, can be achieved usingstandard methods known in the art. As with other undifferentiated orpartially differentiated precursor cells, germline stem cells or theirprogenitors can be induced to follow a particular developmental pathwayby culture in medium containing agents known in the art. Such agents canbe provided through “co-culture” schemes, wherein cells that secretesuch factors are cultured together with germline stem cells or theirprogenitors to direct the development of germline stem cells or theirprogenitors. These agents include, but are not limited to the following(with regards to biological signaling pathways, pharmacological orbiological antagonists, agonists, or other modulators of function are tobe included in each case): Wnt pathway molecules, TGFβ and/or BMPpathway molecules, modulators of epigenetic mechanisms including histonemodification pathways (including but not limited to acetylation,methylation, etc.), gonadotropins, steroid hormones (including but notlimited to estrogen, progesterone, androgens, etc.), IGF and/or insulinsignaling molecules, leptin and related signaling molecules, members ofthe sphingolipid family (including but not limited toSphingosine-1-Phosphate, ceramide, etc.), regulators of apoptosis(including but not limited to Caspase inhibitors, the Bax inhibitorhumanin, etc.), Notch pathway molecules, cell-cycle regulators includingso-called cellular senescence pathways (including but not limited toBmi-1, the Ink4a locus, etc.), regulators of receptor-kinases, andintracellular kinase cascades, and strategies that modulate geneexpression via gene expression interference (including but not limitedto variants of RNA interference, morpholino technologies, or antisenseRNA molecules, etc.). Some specific examples of such factors, theprogenitor/precursor cells on which they act, and the resulting celltypes formed are shown in Table 1.

TABLE 1 Selected Examples of Differentiation Agents AgentProgenitor/precursor Differentiated Cell Vascular Endothelial GrowthEmbryonic Stem Cell Hematopoietic Cell¹ Factor Sonic Hedgehog FloorPlate Motor Neuron² Insulin-like Growth Factor II Embryonic Stem CellMyoblast³ Osteogenin Osteoprogenitor Osteoblast⁴ Cytotoxic T CellDifferentiation Spleen Cell Cytotoxic T Lymyphocyte⁵ Factor □-cateninSkin Stem Cell Follicular Keratinocyte⁶ Bone Morphogenic Protein 2Mesenchymal Stem Cell Adipocytes, Osteoblasts⁷ Interleukin 2 Bone MarrowPrecursor Natural Killer Cells⁸ Transforming Growth Factor □ CardiacFibroblast Cardiac Myocyte⁹ Nerve Growth Factor Chromaffin CellSympathetic Neuron¹⁰ Steel Factor Neural Crest Melanocyte¹¹ Interleukin1 Mesencephalic Progenitor Dopaminergic Neuron¹² Fibroblast GrowthFactor 2 GHFT Lactotrope¹³ Retinoic Acid Promyelocytic LeukemiaGranulocyte¹⁴ Wnt3 Embryonic Stem Cell Hematopoietic Cell¹⁵ ¹Keller, etal. (1999) Exp. Hematol. 27: 777-787. ²Marti, et al. (1995) Nature. 375:322-325. ³Prelle, et al. (2000) Biochem. Biophy. Res. Commun. 277:631-638. ⁴Amedee, et al. (1994) Differentiation. 58: 157-164. ⁵Hardt, etal. (1985) Eur. J. Immunol. 15: 472-478. ⁶Huelsken, et al. (2001) Cell.105: 533-545. ⁷Ji, et al. (2000) J. Bone Miner. Metab. 18: 132-139.⁸Migliorati, et al. (1987) J. Immunol. 138: 3618-3625. ⁹Eghbali, et al.(1991) Proc. Natl. Acad. Sci. USA. 88: 795-799. ¹⁰Niijima, et al. (1995)J. Neurosci. 15: 1180-1194. ¹¹Guo, et al. (1997) Dev. Biol. 184: 61-69.¹²Ling, et al. (1998) Exp. Neurol. 149: 411-423. ¹³Lopez-Fernandez, etal. (2000) J. Biol. Chem. 275: 21653-60. ¹⁴Wang, et al. (1989) Leuk.Res. 13: 1091-1097. ¹⁵Lalco, et al. (2001) Mech. Dev. 103: 49-59.

The cells of the present invention can provide a variety of cell types,including terminally differentiated and undifferentiated cell types, forhigh-throughput screening techniques used to identify a multitude oftarget biologic or pharmacologic agents. Importantly, the femalegermline stem cells or their progenitor cells provide a source ofcultured cells from a variety of genetically diverse subjects, who mayrespond differently to biologic and pharmacologic agents.

The present invention provides methods for using the germline stemcells, or their progenitors, described herein to characterizepharmacogenetic cellular responses to biologic or pharmacologic agents.In the method of using germline stem cells or their progenitors tocharacterize pharmacogenetic cellular responses to biologic orpharmacologic agents, or combinatorial libraries of such agents,germline stem cells or their progenitors are preferably isolated from astatistically significant population of subjects, culture expanded, andcontacted with one or more biologic or pharmacologic agents. Germlinestem cells of the invention or their progenitors optionally can beinduced to differentiate, wherein differentiated cells are the desiredtarget for a certain biologic or pharmacologic agent, either prior to orafter culture expansion. By comparing the one or more cellular responsesof the cultures from subjects in the statistically significantpopulation, the effects of the biologic or pharmacologic agent can bedetermined. Effects of the biologic or pharmacologic agent can beinduction of apoptosis, changes in gene expression, chromosomal damage,and decreases or increases in hormones involved in ovarian function.

Alternatively, genetically identical germline stem cells, theirprogenitors, or their progeny, can be used to screen separate compounds,such as compounds of a combinatorial library. Gene expression systemsfor use in combination with cell-based high-throughput screening havebeen described (Jayawickreme, C. and Kost, T., (1997) Curr. Opin.Biotechnol. 8: 629-634).

The invention also envisions a tissue-engineered organ (e.g., ovary), orportion, or specific section thereof, or a tissue engineered devicecomprising a tissue of interest and optionally, cytokines, hormones,growth factors, or differentiation factors that induce differentiationinto a desired cell type, wherein the germline stem cells of theinvention or their progenitors are used to generate tissues including,but not limited to ovarian tissue. Tissue-engineered organs can be usedwith a biocompatible scaffold to support cell growth in athree-dimensional configuration, which can be biodegradable.Tissue-engineered organs generated from the germline stem cells of thepresent invention or their progenitors can be implanted into a subjectin need of a replacement organ, portion, or specific section thereof.

Homogenous organs, portions, or individual cells derived from thegermline stem cell or progenitor cultures of the invention can beimplanted into a host. Likewise, heterogeneous organs, portions, orsections derived from germline stem cells or their progenitors inducedto differentiate into multiple tissue types can be implanted into asubject in need thereof. The transplantation can be autologous, suchthat the donor of the stem cells from which organ or organ units arederived is the recipient of the engineered tissue. The transplantationcan be heterologous, such that the donor of the stem cells from whichorgan or organ units are derived is not that of the recipient of theengineered-tissue.

Once transferred into a host, the tissue-engineered organs canrecapitulate the function and architecture of the native host tissue.The tissue-engineered organs will benefit subjects in a wide variety ofapplications, including the treatment of cancer and other diseasedisclosed herein, congenital defects, or damage due to surgicalresection.

Polymer scaffolds that can be used in the development oftissue-engineered organs derived from the germline stem cells of theinvention function in place of a connective tissue scaffold or matrix,and are designed to optimize gas, nutrient, and waste exchange bydiffusion. Polymer scaffolds can comprise, for example, a porous,non-woven array of fibers. The polymer scaffold can be shaped tomaximize surface area, to allow adequate diffusion of nutrients andgrowth factors to the cells. Taking these parameters into consideration,one of skill in the art could configure a polymer scaffold havingsufficient surface area for the cells to be nourished by diffusion untilnew blood vessels interdigitate the implanted engineered-tissue usingmethods known in the art. Polymer scaffolds can comprise a fibrillarstructure. The fibers can be round, scalloped, flattened, star-shaped,solitary or entwined with other fibers. Branching fibers can be used,increasing surface area proportionately to volume.

Unless otherwise specified, the term “polymer” includes polymers andmonomers that can be polymerized or adhered to form an integral unit.The polymer can be non-biodegradable or biodegradable, typically viahydrolysis or enzymatic cleavage. The term “biodegradable” refers tomaterials that are bioresorbable and/or degrade and/or break down bymechanical degradation upon interaction with a physiological environmentinto components that are metabolizable or excretable, over a period oftime from minutes to three years, preferably less than one year, whilemaintaining the requisite structural integrity. As used in reference topolymers, the term “degrade” refers to cleavage of the polymer chain,such that the molecular weight stays approximately constant at theoligomer level and particles of polymer remain following degradation.

Materials suitable for polymer scaffold fabrication include polylacticacid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic acid (PDLA),polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide (PLGA),polydioxanone, polygluconate, polylactic acid-polyethylene oxidecopolymers, modified cellulose, collagen, polyhydroxybutyrate,polyhydroxpriopionic acid, polyphosphoester, poly(alpha-hydroxy acid),polycaprolactone, polycarbonates, polyamides, polyanhydrides, polyaminoacids, polyorthoesters, polyacetals, polycyanoacrylates, degradableurethanes, aliphatic polyester polyacrylates, polymethacrylate, acylsubstituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl flouride, polyvinylimidazole, chlorosulphonated polyolifins, polyethylene oxide, polyvinylalcohol, Teflon®, nylon silicon, and shape memory materials, such aspoly(styrene-block-butadiene), polynorbornene, hydrogels, metallicalloys, and oligo(ε-caprolactone)diol as switchingsegment/oligo(p-dioxyanone)diol as physical crosslink. Other suitablepolymers can be obtained by reference to The Polymer Handbook, 3rdedition (Wiley, N.Y., 1989).

Factors, including but not limited to nutrients, growth factors,inducers of differentiation or de-differentiation, products ofsecretion, immunomodulators, inhibitors of inflammation, regressionfactors, hormones, or other biologically active compounds can beincorporated into or can be provided in conjunction with the polymerscaffold.

IV. Screening Assays

The invention provides methods for identifying modulators, i.e.,candidate or test compounds or agents (e.g., proteins, peptides,peptidomimetics, peptoids, small molecules or other drugs) whichmodulate female germline stem cells or female germline stem cellprogenitor cells. Agents thus identified can be used to modulate, forexample, proliferation, survival and differentiation of a femalegermline stem cell or its progenitor e.g., in a therapeutic protocol.

The test agents of the present invention can be obtained singly or usingany of the numerous approaches in combinatorial library methods known inthe art, including: biological libraries; peptoid libraries (librariesof molecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann, R. N. (1994)et al., J. Med. Chem. 37:2678-85); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992), Biotechniques 13:412-421), or on beads (Lam (1991), Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al. (1992) Proc Natl. Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

Chemical compounds to be used as test agents (i.e., potential inhibitor,antagonist, agonist) can be obtained from commercial sources or can besynthesized from readily available starting materials using standardsynthetic techniques and methodologies known to those of ordinary skillin the art. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds identified by the methods described herein are known in theart and include, for example, those such as described in R. Larock(1989) Comprehensive Organic Transformations, VCH Publishers; T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nded., John Wiley and Sons (1991); L. Fieser And M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995), and subsequent editions thereof.

In one aspect the compounds are organic small molecules, that is,compounds having molecular weight less than 1,000 amu, alternativelybetween 350-750 amu. In other aspects, the compounds are: (i) those thatare non-peptidic; (ii) those having between 1 and 5, inclusive,heterocyclyl, or heteroaryl ring groups, which may bear furthersubstituents; (iii) those in their respective pharmaceuticallyacceptable salt forms; or (iv) those that are peptidic.

The term “heterocyclyl” refers to a nonaromatic 3-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring can be substituted by a substituent.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring can be substituted by a substituent.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of thatgroup. Suitable substituents include, without limitation, alkyl,alkenyl, allynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H,perfluoroalkyl, perfiuoroalkoxy, methylenedioxy, ethylenedioxy,carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(O)_(n)alkyl(where n is 0-2), S(O)_(n) aryl (where n is 0-2), S(O)_(n) heteroaryl(where n is 0-2), S(O)_(n) heterocyclyl (where n is 0-2), amine(mono-,di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinationsthereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-,alkyl, aralkyl, heteroaralkyl, and combinations thereof),sulfonamide(mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinationsthereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstitutedheterocyclyl, and unsubstituted cycloalkyl. In one aspect, thesubstituents on a group are independently any one single, or any subsetof the aforementioned substituents.

Combinations of substituents and variables in compounds envisioned bythis invention are only those that result in the formation of stablecompounds. The term “stable”, as used herein, refers to compounds whichpossess stability sufficient to allow manufacture and which maintainsthe integrity of the compound for a sufficient period of time to beuseful for the purposes detailed herein (e.g., transport, storage,assaying, therapeutic administration to a subject).

The compounds described herein can contain one or more asymmetriccenters and thus occur as racemates and racemic mixtures, singleenantiomers, individual diastereomers and diastereomeric mixtures. Allsuch isomeric forms of these compounds are expressly included in thepresent invention. The compounds described herein can also berepresented in multiple tautomeric forms, all of which are includedherein. The compounds can also occur in cis- or trans- or E- or Z-doublebond isomeric forms. All such isomeric forms of such compounds areexpressly included in the present invention.

Test agents of the invention can also be peptides (e.g., growth factors,cytokines, receptor ligants).

Screening methods of the invention can involve the identification of anagent that increases the proliferation or survival of female germlinestem cells or female germline stem cell progenitor cells. Such methodswill typically involve contacting a population of the female germlinestem or progenitor cells with a test agent in culture and quantitatingthe number of new stem or progenitor cells produced as a result.Comparison to an untreated control can be concurrently assessed. Wherean increase in the number of stem or progenitor cells is detectedrelative to the control, the test agent is determined to have thedesired activity.

In practicing the methods of the invention, it may be desirable toemploy a purified population of female germline stem cells or theirprogenitor cells. A purified population of female germline stem cells orfemale germline stem cell progenitor cells have about 50-55%, 55-60%,60-65% and 65-70% purity. More preferably the purity is about 70-75%,75-80%, 80-85%; and still more preferably the purity is about 85-90%,90-95%, and 95-100%.

In other methods, the test agent is assayed using a biological samplerather than a purified population of stem or progenitor cells. The term“biological sample” includes tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. Preferred biological samples include bone marrow,peripheral blood and ovarian tissue.

Increased amounts of female germline stem cells or female germline stemcell progenitor cells can also be detected by an increase in geneexpression of genetic markers including an SSEA (e.g., SSEA-1), Oct-4,Dazl, Stella and Vasa. The level of expression can be measured in anumber of ways, including, but not limited to: measuring the mRNAencoded by the genetic markers; measuring the amount of protein encodedby the genetic markers; or measuring the activity of the protein encodedby the genetic markers.

The level of mRNA corresponding to a genetic marker can be determinedboth by in situ and by in vitro formats. The isolated mRNA can be usedin hybridization or amplification assays that include, but are notlimited to, Southern or Northern analyses, polymerase chain reactionanalyses and probe arrays. One diagnostic method for the detection ofmRNA levels involves contacting the isolated mRNA with a nucleic acidmolecule (probe) that can hybridize to the mRNA encoded by the genebeing detected. The nucleic acid probe is sufficient to specificallyhybridize under stringent conditions to mRNA or genomic DNA. The probecan be disposed on an address of an array, e.g., an array describedbelow. Other suitable probes for use in the diagnostic assays aredescribed herein.

In one format, mRNA (or cDNA) is immobilized on a surface and contactedwith the probes, for example by running the isolated mRNA on an agarosegel and transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probes are immobilized ona surface and the mRNA (or cDNA) is contacted with the probes, forexample, in a two-dimensional gene chip array described below. A skilledartisan can adapt known mRNA detection methods for use in detecting thelevel of mRNA encoded by the genetic markers described herein.

The level of mRNA in a sample can be evaluated with nucleic acidamplification, e.g., by rtPCR (Mullis (1987) U.S. Pat. No. 4,683,202),ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA88:189-193), self sustained sequence replication (Guatelli et al. (1990)Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplificationsystem (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177),Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rollingcircle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or anyother nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques known in the art. As usedherein, amplification primers are defined as being a pair of nucleicacid molecules that can anneal to 5′ or 3′ regions of a gene (plus andminus strands, respectively, or vice-versa) and contain a short regionin between. In general, amplification primers are from about 10 to 30nucleotides in length and flank a region from about 50 to 200nucleotides in length. Under appropriate conditions and with appropriatereagents, such primers permit the amplification of a nucleic acidmolecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, a cell or tissue sample can be prepared/processedand immobilized on a support, typically a glass slide, and thencontacted with a probe that can hybridize to mRNA that encodes thegenetic marker being analyzed.

Screening methods of the invention can involve the identification of anagent that increases the differentiation of female germline stem cellsor female germline stem cell progenitor cells into oocytes. Such methodswill typically involve contacting a population of the stem or progenitorcells with a test agent in culture and quantitating the number of newoocytes produced as a result. Comparison to an untreated control can beconcurrently assessed. Where an increase in the number of oocytes isdetected relative to the control, the test agent is determined to havethe desired activity. The test agent can also be assayed using abiological sample (e.g., ovarian tissue); subsequent testing using apopulation of stem or progenitor cells may be conducted to distinguishthe functional activity of the agent (e.g., differentiation rather thenincrease in proliferation or survival) where the result is ambiguous.

Increased amounts of oocytes can be detected by a decrease in geneexpression of stem or progenitor genetic markers including an SSEA(e.g., SSEA-1), Oct-4, Dazl, Stella and Vasa or an increase in oocytemarkers, such as HDAC6, GDF9 and ZP3.

Screening methods of the invention can involve the identification of anagent that decreases the proliferation or survival of female germlinestem cells or female germline stem cell progenitor cells. Such methodswill typically involve contacting a population of the stem or progenitorcells, or a biological sample containing said cells with a test agent inculture and quantitating the number of stem or progenitor cells lost asa result. Comparison to an untreated control can be concurrentlyassessed. Where a decrease in the number of stem or progenitor cells isdetected relative to the control, the test agent is determined to havethe desired activity.

IV. Methods of Treatment

Female germline stem cells of the invention or their progenitors can beused in a variety of therapeutic applications (e.g., oocyte generationfor in vivo restoration or ex vivo procedures including in vitrofertilization and somatic cell nuclear transfer). Accordingly, methodsof the invention relate to the use of female germline stem cells, orprogenitors derived from female germline stem cells, to, among otherthings, expand the follicle reserve as a means of enhancing or restoringfertility in females, and for ameliorating symptoms and consequences ofmenopause.

Thus, the present invention provides methods for treating infertilitycomprising providing a female germline stem cell, its progenitor, or theprogeny thereof, to a female subject in need thereof, wherein the cellengrafts into a tissue and differentiates into an oocyte, which canlater be provided for fertilization (e.g., following ovulation or invitro fertilization). Preferably, the tissue is ovarian tissue, however,other tissues in the body may host the engrafted cell that in turngenerates an oocyte. Oocytes harbored in extra-ovarian tissues can beharvested and used for procedures including in vitro fertilization.

The present invention also provides methods for treating infertilitycomprising contacting ovarian tissue of a female subject in need thereofwith an agent that increases the production or survival of femalegermline stem cells or their progenitors. As previously described,oocyte production can be increased by increasing the number (i.e.,proliferation) or life span (i.e., survival) of female germline stemcells or their progenitors, as well as by differentiating femalegermline stem cells or their progenitors into oocytes. Such oocytes canlater be provided for fertilization following ovulation in the subject.

The present invention also provides methods for repairing damagedovarian tissue, comprising providing a female germline stem cell, or itsprogenitor, to the tissue, wherein the cell engrafts into the tissue anddifferentiates into an oocyte. Damage can be caused, for example, byexposure to cytotoxic factors, chemotherapeutic drugs, radiation,hormone deprivation, growth factor deprivation, cytokine deprivation,cell receptor antibodies, and the like. Chemotherapeutic drugs include,but are not limited to, 5-FU, vinblastine, actinomycin D, etoposide,cisplatin, methotrexate, doxorubicin, among others. Damage can also becaused be diseases that affect ovarian function, including, but notlimited to cancer, polycystic ovary disease, genetic disorders, immunedisorders, metabolic disorders, and the like.

The present invention also provides methods for restoring ovarianfunction in a menopausal female subject, comprising providing a femalegermline stem cell, or its progenitor, to the subject, wherein the cellengrafts into the ovary and differentiates into an oocyte. Themenopausal female subject can be in a stage of either peri- orpost-menopause, with said menopause caused by either normal (e.g.,aging) or pathological (e.g., surgery, disease, ovarian damage)processes.

Ovarian function in a post-menopausal female can also be restored bycontacting ovarian tissue of the subject with an agent that increasesthe amount of female germline stem cells or their progenitors (e.g., byincreasing the number or life span of female germline stem cells, aswell as by increasing the differentiation of female germline stem cellsinto oocytes).

Restoration of ovarian function can relieve adverse symptoms andcomplications associated with menopausal disorders, including, but notlimited to, somatic disorders such as osteoporosis, cardiovasculardisease, somatic sexual dysfunction, hot flashes, vaginal drying, sleepdisorders, depression, irritability, loss of libido, hormone imbalances,and the like, as well as cognitive disorders, such as loss of memory;emotional disorders, depression, and the like.

The present invention further provides a method for contraception in afemale subject comprising contacting ovarian tissue of the subject withan agent that decreases the proliferation, function or survival offemale germline stem cells or their progenitors.

Germline stem cells of the invention, their progenitors or their invitro-derived progeny, can be administered as previously described.Prior to administration, germline stem cells, their progenitors or theirin vitro-derived progeny, described herein can optionally be geneticallymodified, in vitro, in vivo or ex vivo, by introducing heterologous DNAor RNA or protein into the cell by a variety of recombinant methodsknown to those of skill in the art. These methods are generally groupedinto four major categories: (1) viral transfer, including the use of DNAor RNA viral vectors, such as retroviruses (including lentiviruses),Simian virus 40 (SV40), adenovirus, Sindbis virus, and bovinepapillomavirus, for example; (2) chemical transfer, including calciumphosphate transfection and DEAE dextran transfection methods; (3)membrane fusion transfer, using DNA-loaded membranous vesicles such asliposomes, red blood cell ghosts, and protoplasts, for example; and (4)physical transfer techniques, such as microinjection, electroporation,or direct “naked” DNA transfer.

The germline stem cells of the invention, their progenitors or their invitro-derived progeny, can be genetically altered by insertion ofpre-selected isolated DNA, by substitution of a segment of the cellulargenome with pre-selected isolated DNA, or by deletion of or inactivationof at least a portion of the cellular genome of the cell. Deletion orinactivation of at least a portion of the cellular genome can beaccomplished by a variety of means, including but not limited to geneticrecombination, by antisense technology (which can include the use ofpeptide nucleic acids, or PNAs), or by ribozyme technology, for example.The altered genome may contain the genetic sequence of a selectable orscreenable marker gene that is expressed so that the cell with alteredgenome, or its progeny, can be differentiated from cells having anunaltered genome. For example, the marker may be a green, red, yellowfluorescent protein, β-galactosidase, the neomycin resistance gene,dihydrofolate reductase (DHFR), or hygromycin, but are not limited tothese examples.

In some cases, the underlying defect of a pathological state is amutation in DNA encoding a protein such as a metabolic protein.Preferably, the polypeptide encoded by the heterologous DNA lacks amutation associated with a pathological state. In other cases, apathological state is associated with a decrease in expression of aprotein. A genetically altered germline stem cell, or its progeny, maycontain DNA encoding such a protein under the control of a promoter thatdirects strong expression of the recombinant protein. Alternatively, thecell may express a gene that can be regulated by an inducible promoteror other control mechanism where conditions necessitate highlycontrolled regulation or timing of the expression of a protein, enzyme,or other cell product. Such stem cells, when transplanted into a subjectsuffering from abnormally low expression of the protein, produce highlevels of the protein to confer a therapeutic benefit. For example, thegermline stem cell of the invention, its progenitors or its invitro-derived progeny, can contain heterologous DNA encoding genes to beexpressed, for example, in gene therapy. Germline stem cells of theinvention, their progenitors or their progeny, can contain heterologousDNA encoding Atm, the gene responsible for the human diseaseAtaxia-telangiectasia in which fertility is disrupted. Providing Atm viagermline stem cells, its progenitors or its in vitro-derived progeny,can further relieve defects in ovarian function. DNA encoding a geneproduct that alters the functional properties of germline stem cells inthe absence of any disease state is also envisioned. For example,delivery of a gene that inhibits apoptosis, or that preventsdifferentiation would be beneficial.

Insertion of one or more pre-selected DNA sequences can be accomplishedby homologous recombination or by viral integration into the host cellgenome. The desired gene sequence can also be incorporated into thecell, particularly into its nucleus, using a plasmid expression vectorand a nuclear localization sequence. Methods for directingpolynucleotides to the nucleus have been described in the art. Thegenetic material can be introduced using promoters that will allow forthe gene of interest to be positively or negatively induced usingcertain chemicals/drugs, to be eliminated following administration of agiven drug/chemical, or can be tagged to allow induction by chemicals(including but not limited to the tamoxifen responsive mutated estrogenreceptor) expression in specific cell compartments (including but notlimited to the cell membrane).

Calcium phosphate transfection can be used to introduce plasmid DNAcontaining a target gene or polynucleotide into isolated or culturedgermline stem cells or their progenitors and is a standard method of DNAtransfer to those of skill in the art. DEAE-dextran transfection, whichis also known to those of skill in the art, may be preferred overcalcium phosphate transfection where transient transfection is desired,as it is often more efficient. Since the cells of the present inventionare isolated cells, microinjection can be particularly effective fortransferring genetic material into the cells. This method isadvantageous because it provides delivery of the desired geneticmaterial directly to the nucleus, avoiding both cytoplasmic andlysosomal degradation of the injected polynucleotide. This technique hasbeen used effectively to accomplish germline modification in transgenicanimals. Cells of the present invention can also be genetically modifiedusing electroporation.

Liposomal delivery of DNA or RNA to genetically modify the cells can beperformed using cationic liposomes, which form a stable complex with thepolynucleotide. For stabilization of the liposome complex, dioleoylphosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPA)can be added. Commercially available reagents for liposomal transferinclude Lipofectin (Life Technologies). Lipofectin, for example, is amixture of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-N—N—N-trimethyl ammonia chloride and DOPE.Liposomes can carry larger pieces of DNA, can generally protect thepolynucleotide from degradation, and can be targeted to specific cellsor tissues. Cationic lipid-mediated gene transfer efficiency can beenhanced by incorporating purified viral or cellular envelopecomponents, such as the purified G glycoprotein of the vesicularstomatitis virus envelope (VSV-G). Gene transfer techniques which havebeen shown effective for delivery of DNA into primary and establishedmammalian cell lines using lipopolyamine-coated DNA can be used tointroduce target DNA into the germline stem cells described herein.

Naked plasmid DNA can be injected directly into a tissue mass formed ofdifferentiated cells from the isolated germline stem cells or theirprogenitors. This technique has been shown to be effective intransferring plasmid DNA to skeletal muscle tissue, where expression inmouse skeletal muscle has been observed for more than 19 monthsfollowing a single intramuscular injection. More rapidly dividing cellstake up naked plasmid DNA more efficiently. Therefore, it isadvantageous to stimulate cell division prior to treatment with plasmidDNA. Microprojectile gene transfer can also be used to transfer genesinto stem cells either in vitro or in vivo. The basic procedure formicroprojectile gene transfer was described by J. Wolff in GeneTherapeutics (1994), page 195. Similarly, microparticle injectiontechniques have been described previously, and methods are known tothose of skill in the art. Signal peptides can be also attached toplasmid DNA to direct the DNA to the nucleus for more efficientexpression.

Viral vectors are used to genetically alter germline stem cells of thepresent invention and their progeny. Viral vectors are used, as are thephysical methods previously described, to deliver one or more targetgenes, polynucleotides, antisense molecules, or ribozyme sequences, forexample, into the cells. Viral vectors and methods for using them todeliver DNA to cells are well known to those of skill in the art.Examples of viral vectors that can be used to genetically alter thecells of the present invention include, but are not limited to,adenoviral vectors, adeno-associated viral vectors, retroviral vectors(including lentiviral vectors), alphaviral vectors (e.g., Sindbisvectors), and herpes virus vectors.

Peptide or protein transfection is another method that can be used togenetically alter germline stem cells of the invention and theirprogeny. Peptides including, but not limited to, Pep-1 (commerciallyavailable as Chariot™) and MPG, can quickly and efficiently transportbiologically active proteins, peptides, antibodies, and nucleic acidsdirectly into cells, with an efficiency of about 60% to about 95%(Morris, M. C. et al, (2001) Nat. Biotech. 19: 1173-1176). Withoutwishing to be bound by theory, the peptide forms a non-covalent bondwith the macromolecule of interest (i.e., protein, nucleic acid). Thebinding reaction stabilizes the protein and protects it fromdegradation. Upon delivery into the cell of interest, such as stem cellsof the invention, the peptide-macromolecule complex dissociates, leavingthe macromolecule biologically active and free to proceed to its targetorganelle. Delivery can occur in the presence of absence of serum.Uptake and delivery can occur at 4° C., which eliminates endosomalprocessing of incoming macromolecules. Movement of macromoleculesthrough the endosomal pathway can modify the macromolecule upon uptake.Peptides such as Pep-1, by directly delivering a protein, antibody, orpeptide of interest, bypass the transcription-translation process.

Methods of the invention can provide oocyte reserves for use in ex vivoprocedures, such as somatic cell nuclear transfer. Employing recombinanttechniques prior to nuclear transfer will allow for the design ofcustomized oocytes and ultimately produce embryos from which embryonicstem cells can be derived. In addition, genetic manipulation of donorDNA prior to nuclear transfer will result in embryos that possess thedesired modification or genetic trait.

Methods of somatic cell nuclear transfer are well known in the art. SeeU.S. application Ser. No. 10/494,074, filed on Mar. 24, 2004 andpublished as 20050064586; Wilmut et al. (1997) Nature, 385, 810-813;Wakayama, et al. (1998) Nature 394: 369-374; and Teruhiko et al., (1999)PNAS 96:14984-14989. Nuclear transplantation involves thetransplantation of donor cells or cell nuclei into enucleated oocytes.Enucleation of the oocyte can be performed in a number of manners wellknown to those of ordinary skill in the art. Insertion of the donor cellor nucleus into the enucleated oocyte to form a reconstituted cell isusually by microinjection of a donor cell tinder the zona pellucidaprior to fusion. Fusion may be induced by application of a DC electricalpulse across the contact/fusion plane (electrofusion), by exposure ofthe cells to fusion-promoting chemicals, such as polyethylene glycol, orby way of an inactivated virus, such as the Sendai virus. Areconstituted cell is typically activated by electrical and/ornon-electrical means before, during, and/or after fusion of the nucleardonor and recipient oocyte. Activation methods include electric pulses,chemically induced shock, penetration by sperm, increasing levels ofdivalent cations in the oocyte, and reducing phosphorylation of cellularproteins (as by way of kinase inhibitors) in the oocyte. The activatedreconstituted cells, or embryos, are typically cultured in medium wellknown to those of ordinary skill in the art and then transferred to thewomb of an animal.

Methods for the generation of embryonic stem cells from embryos are alsowell known in the art. See Evans, et al. (1981) Nature, 29:154-156;Martin, et al. (1981) PNAS, 78:7634-7638; Smith, et al. (1987)Development Biology, 121:1-9; Notarianni, et al. (1991) J. Reprod.Fert., Suppl. 43:255-260; Chen R L, et al. (1997) Biology ofReproduction, 57 (4):756-764; Wianny, et al. (1999) Theriogenology, 52(2):195-212; Stekelenburg-Hamers, et al. (1995) Mol. Reprod. 40:444-454;Thomson, et al. (1995) PNAS, 92 (17):7844-8 and Thomson (1998) Science,282 (6):1145-1147. Accordingly, embryos produced from oocytes of theinvention can be genetically modified, either through manipulation ofthe oocyte in vitro prior to fertilization or manipulation of donor DNAprior to nuclear transfer into the enucleated oocyte, to produce embryoshaving a desired genetic trait.

VI. In Vitro Fertilization

Oocytes produced from germline stem cells of the invention, orprogenitors derived from germline stem cells of the invention, asdescribed herein may also be used for methods of in vitro fertilization.Accordingly, the invention provides methods for in vitro fertilizationof a female subject. The method comprises the steps of: either producingan oocyte by culturing a female germline stem cell, or its progenitor,in the presence of an oocyte differentiation agent or in vivodifferentiating the female germline stem cell, or its progenitor into anoocyte and obtaining the oocyte; fertilizing the oocyte in vitro to forma zygote; and implanting the zygote into the uterus of a female subject.

Methods of in vitro fertilization are well known in the art, and are nowrapidly becoming commonplace. Couples are generally first evaluated todiagnose their particular infertility problem(s). These may range fromunexplained infertility of both partners to severe problems of thefemale (e.g., endometriosis resulting in nonpatent oviducts withirregular menstrual cycles or polycystic ovarian disease) or the male(e.g., low sperm count with morphological abnormalities, or an inabilityto ejaculate normally as with spinal cord lesions, retrogradeejaculation, or reversed vasectomy). The results of these evaluationsalso determine the specific procedure to be performed for each couple.

Procedures often begin with the administration of a drug todown-regulate the hypothalamic/pituitary system (LHRH agonist). Thisprocess decreases serum concentrations of the gonadotropins, anddeveloping ovarian follicles degenerate, thereby providing a set of newfollicles at earlier stages of development. This permits more precisecontrol of the maturation of these new follicles by administration ofexogenous gonadotropins in the absence of influences by the hypothalamicpituitary axis. The progress of maturation and the number of growingfollicles (usually four to ten stimulated per ovary) are monitored bydaily observations using ultrasound and serum estradiol determinations.When the follicles attain preovulatory size (18-21 mm) and estradiolconcentrations continue to rise linearly, the ovulatory response isinitiated by exogenous administration of human chorionic gonadotropins(hCG).

Oocytes can be obtained from germline stem cells, or progenitors derivedfrom germline stem cells, as previously described herein. Germline stemcells, or progenitors derived from germline stem cells, can be culturedin the presence of an oocyte differentiation agent which inducesdifferentiation into oocytes. The differentiation agent can be suppliedexogenously (e.g., added to the culture medium) or from endogenoussources during co-culture with allogenic or heterogenic ovarian tissue.Female germline stem cells or their progenitors can also be cultured ina tissue-engineered structure wherein the differentiation agent iseither exogenously or endogenously supplied and oocytes are obtained.

Individual oocytes can be evaluated morphologically and transferred to apetri dish containing culture media and heat-inactivated serum. A semensample is provided by the male partner and processed using a “swim up”procedure, whereby the most active, motile sperm will be obtained forinsemination. If the female's oviducts are present, a procedure calledGIFT (gamete intrafallopian transfer) can be performed at this time. Bythis approach, oocyte-cumulus complexes surrounded by sperm are placeddirectly into the oviducts by laproscopy. This procedure best simulatesthe normal sequences of events and permits fertilization to occur withinthe oviducts. Not surprisingly, GIFT has the highest success rate with22% of the 3,750 patients undergoing ova retrieval in 1990 having a livedelivery. An alternative procedure ZIFT (zygote intrafallopian transfer)permits the selection of in vitro fertilized zygotes to be transferredto oviducts the day following ova retrieval. Extra zygotes can becryopreserved at this time for future transfer or for donation tocouples without female gametes. Most patients having more seriousinfertility problems, however, will require an additional one to twodays incubation in culture so that preembryos in the early cleavagestates can be selected for transfer to the uterus. This IVF-UT (in vitrofertilization uterine transfer) procedure entails the transcervicaltransfer of several 2-6 cell (day 2) or 8-16 (day 3) preembryos to thefundus of the uterus (4-5 preembryos provides optimal success).

Procedures for in vitro fertilization are also described in U.S. Pat.Nos., 6,610,543 6,585,982, 6,544,166, 6,352,997, 6,281,013, 6,196,965,6,130,086, 6,110,741, 6,040,340, 6,011,015, 6,010,448, 5,961,444,5,882,928, 5,827,174, 5,760,024, 5,744,366, 5,635,366, 5,691,194,5,627,066, 5,563,059, 5,541,081, 5,538,948, 5,532,155, 5,512,476,5,360,389, 5,296,375, 5,160,312, 5,147,315, 5,084,004, 4,902,286,4,865,589, 4,846,785, 4,845,077, 4,832,681, 4,790,814, 4,725,579,4,701,161, 4,654,025, 4,642,094, 4,589,402, 4,339,434, 4,326,505,4,193,392, 4,062,942, and 3,854,470, the contents of which arespecifically incorporated by reference for their description of theseprocedures.

The following examples are put forth for illustrative purposes only andare not intended to limit the scope of what the inventors regard astheir invention.

EXAMPLES Example 1 Post-Natal Ovarian Germ-Cell Dynamics

Counts of healthy (non-atretic) and degenerating (atretic) follicles inovaries of female mice were made to assess germ-cell dynamics in femalemammals. In particular, degeneration rates were calculated, to determinethe predictive age at which exhaustion of the oocyte (i.e., follicle)reserve would occur in the absence of new oocyte production.

Age-specified or timed-pregnant wild-type C57BL/6 and CD1 female micewere obtained from Charles River Laboratories, whereas AKR/J mice wereobtained from Jackson Laboratories. Ovaries were fixed in 0.34N glacialacetic acid, 10% formalin, and 28% ethanol, paraffin embedded, andserially sectioned (8 μm). The sections were aligned in order on glassmicroscope slides, and stained with hematoxylin and picric methyl blue.The number of non-atretic or atretic primordial, primary, and preantralfollicles was then determined. Primordial follicles were identified ashaving a compact oocyte surrounded by a single layer of flattenedgranulosa cells, while primary follicles were identified as having anenlarged oocyte surrounded by a single layer of cuboidal granulosacells. Intermediate-stage follicles (compact or enlarged oocyte with asingle layer of mixed flattened and cuboidal granulosa cells) werescored as primary. Preantral follicles were identified as having anenlarged oocyte surrounded by at least a partial or complete secondlayer of cuboidal granulosa cells, but no more than four complete layersof cuboidal granulosa cells. Only those follicles containing an oocytewith a clearly visible nucleus were scored. Follicles at the primordial,primary and preantral (immature) stages of development were scored asatretic if the oocyte was degenerating (convoluted, condensed) orfragmented. Grossly atretic follicles lacking oocyte remnants were notincluded in the analyses. Given that this procedure sampled one-fifth ofthe entire ovarian volume, the total number of follicles per ovary(healthy or atretic) was then estimated by multiplying the cumulativecounts for each ovary by a correction factor of five (Zuckerman, S.(1951) Recent Prog. Horm. Res. 6: 63-108; Tilly, J. L. (2003) Reprod.Biol. Endocrinol. 1:1-11). A single trained ovarian histologist in ablinded fashion conducted all counts, and two other members of the groupperiodically evaluated random samples to verify accuracy andreproducibility of the data.

Analysis of non-atretic quiescent (primordial) and early growing(primary, preantral) follicle numbers revealed that approximatelyone-third of the peak endowment of immature follicles was lost duringdevelopment to young adulthood (see FIG. 1 a), consistent with previousstudies of follicle development in mice. Through the first 20 days ofage, atresia occurred at a low but constant rate (FIG. 1 b), consistentwith a proportional decline in non-atretic follicle numbers during thistime period (FIG. 1 a). However, the incidence of atresia increasedmarkedly by day 30 and further by day 40, reaching a peak level of morethan 1,200 dying follicles per ovary on day 42 that was maintained wellinto reproductive life (FIG. 1 b).

Clearance of apoptotic cells in vivo occurs within 3-18 hours (Wyllie,A. H. et al, (1980) Int. Rev. Cytol. 68: 251-306; Uri, K. and Potten, C.S. (1983) Br. J. Cancer 47: 175-185; Bursch, W. et al, (1990)Carcinogenesis 11: 847-853). Nonetheless, experiments were conducted torule out the possibility that the large atretic follicle populationobserved in adult animals simply represented accumulation of oocytecorpses in follicles that had degenerated weeks earlier. The firstexperiment, based on past studies demonstrating that extensive levels ofoocyte apoptosis occur in the newborn mouse ovary coincident withfollicle formation, evaluated changes in the number of non-atreticoocytes between days 1 and 4 postpartum compared with the number ofdegenerative oocytes on day 4. More than 8,000 non-atretic oocytes werepresent per ovary on day 1, and this pool was reduced by almost 50% byday 4. However, only 218 degenerative oocytes per ovary were found onday 4, indicating that over 3,000 oocytes had died and had been clearedfrom the ovary between days 1 and 4 postpartum. Table 2 contains thedata measuring developmental degeneration and clearance of oocytes inthe neonatal mouse ovary.

TABLE 2 Developmental degeneration and clearance of oocytes in neonatalmouse ovaries Endpoint Analyzed Age(s) postpartum Number(s) per ovaryNon-atretic oocytes Day 1 8,338 ± 1,150 Non-atretic oocytes Day 4 4,733± 68   Oocytes lost Day 1 to Day 4 3605 Atretic oocytes Day 4 218 ± 26 Atretic oocytes cleared Day 1 to Day 4 3,387

A second approach to assessing clearance rates of degenerative oocyteswas employed, using the chemical 9,10-dimethylbenz[a]anthracene (DMBA)to synchronize primordial and primary follicle atesia. Past studies haveshown that DMBA induces degeneration of immature oocytes in a mannerthat morphologically resembles developmental oocyte death. For theseexperiments, C57BL/6 mice were given a single intraperitoneal injectionof vehicle (corn oil) or DMBA (80 mg per kg body weight; resuspended incorn oil) on day 25 postpartum, and ovaries were collected just beforeinjection and at 24-hour intervals after injection for up to 96 hours.In female mice given a single injection of DMBA, the incidence offollicle atresia increased markedly between 24 and 48 hours afterinjection, and remained at a plateau of approximately 850 atreticfollicles per ovary from 48 to 72 hours after injection (FIG. 1 c). By96 hours after injection, there were no healthy primordial or primaryfollicles remaining in the ovaries, and the incidence of atresiareturned to near-basal levels (FIG. 1 c). Therefore, similar to theclearance rate of degenerative oocytes between postnatal days 1 and 4,DMBA-induced synchronization of atresia revealed that over 3,500 oocytescontained within primordial and primary follicles initiated apoptosisand were cleared from the ovary within a 3-day period.

Given this finding, which is that from 1% (days 8, 12, and 20) to asmuch as 16% (day 40) or more (33%, day 42; see FIG. 1 d) of the immaturefollicle pool is degenerating at any given time under normal conditions,complete exhaustion of the follicle reserve by young adulthood would bepredicted. However, the non-atretic pool of follicles declined from peaklevels on day 12 by only 36% on day 40 (FIG. 1 a). This indicated thatthe rate of follicle depletion during post-natal life, as determined byassessing changes in non-atretic follicle numbers, was highlyincongruous with the numbers of follicles actually being eliminated fromthe ovaries through atresia in the same time frame.

To confirm that these findings were not a phenomenon related to C57BL/6mice, changes in follicle numbers from birth to adulthood were analyzedin other strains of mice and compared with corresponding data fromC57BL/6 females. In CD1 mice, the non-atretic follicle pool declined byonly 4% between days 4 and 42 postpartum, despite a relatively highincidence of atresia, comparable to that observed in C57BL/6 females(FIG. 1 d). The non-atretic follicle population in AKR/J mice was 20%larger on day 42 than on day 4, again despite a marked incidence ofatresia (FIG. 1 d). These data highlight a clear discordance betweenchanges in non-atretic follicle numbers and the corresponding incidenceof atresia in the postnatal mammalian ovary.

Similar studies were conducted in rhesus monkeys. Previous analyses ofpostnatal oocyte loss had been explained by simple exponential decay(Olesen, C. et al, (2004) Mol. Reprod. Dev. 67(1): 116-26). Oocyte lossafter a number of periods of time had been projected by utilizing thefollowing equation: t_(n)=a(1−r)^(n); where t_(n)=the calculated numberof remaining oocytes after n periods of time and a constant percentageof dying follicles, represented by r. Results from applying thisequation using the numbers of healthy and dying follicles in the ovariesof rhesus monkeys were inconsistent with the idea that no new folliclesare formed after birth in the primate. Rather, these data argue thatfemale primates, like female mice, must produce new oocytes duringadolescence and adult life.

Using ovaries from adolescent and mature female rhesus monkeys,Vemande-Van Eck measured both the percentage of dying oocytes at anygiven time (4.5%) as well as the rate of clearance of dying oocytes fromthe ovaries (14 days maximum). Using these values, exponential decay ofoocytes would result in an approximate 95% reduction of the oocyte poolin only two years of time (see FIG. 2). The incidence of dying follicleswas stable in both juvenile and in adult life. Thus, regardless of theage at which such decay is initiated, the rhesus monkey ovary would bein danger of entering a menopausal state in only two years. However, therhesus monkey ovary is known to function from the onset of puberty (atapproximately 4 years of age) for about 20 years prior to the onset ofmenopause (Schramm, R. D. et al, (2002) Hum. Reprod. 17: 1597-1603).However, projecting the exponential decay curve given Vermande Van-Eck'sparameters to 7.7 years results in only 6 remaining oocytes.

As with the experiments conduced in mice, the findings in rhesus monkeysare incompatible with the concept of a fixed pool of oocytes at birth infemale primates. The model described herein depicts a mechanism forcontinuing post-natal ovarian follicle renewal.

Example 2 Expression of Meiotic Entry Genes and Genes Implicated inStem-Cell Function in Post-Natal Ovaries

Replication of germ cells to produce oocytes for follicle formation inpostnatal life would require expression of genes involved in theinitiation of meiosis. Thus, expression of synaptonemal complex protein3 (SCP3), a meiosis-specific protein necessary for formation of axiallateral elements of the synaptonemal complex, was examined in juvenileand young adult mouse ovaries. After fixation in 4% neutral-bufferedparaformaldehyde and embedding in paraffin, 6-μm tissue sections werecut from the ovaries and mounted on slides. The sections were de-waxedin xylenes, re-hydrated, and boiled for 5 minutes in 10 mM sodiumcitrate using a microwave.

Primary antibodies specific for SCP3 were then used forimmunohistochemical analyses per the supplier's recommendations. Normaldonkey serum was used in the TNK solution for blocking, and a 1:300dilution of a goat anti-SCP3 antibody (Walpita, D. et al, (1999) Proc.Natl. Acad. Sci. 96: 5622-5627; Russell, L. B. et al, (2000) Mutat. Res.464: 201-212) was applied to the sections followed by a biotinylateddonkey anti-goat IgG (Santa Cruz Biotechnology) for detection using thestreptavidin-peroxidase conjugate system with diaminobenzidine as thecolorimetric substrate. To prevent masking of the immunoreactive signalwith vital dyes, photomicrographs of the sections were taken underHoffman optics without prior counterstaining. Immunohistochemicallocalization of SCP3 revealed individual immunoreactive cells in orproximal to the surface of the ovary (FIGS. 3 a and 3 b). Thepossibility that SCP3 was simply carried over as a stable proteinproduct in oocytes formed during the perinatal period was ruled out bythe finding that oocytes contained within immature follicles were notimmunoreactive (FIGS. 3 c and 3 d).

Postnatal ovarian expression of SCP3 was confirmed at the messenger RNAlevel, as was expression of the endonuclease SPO11 and the recombinaseDMC1 (FIG. 3 e), both of which are also required for the initiation ofmeiosis in mammals. Additionally, expression levels of genes relating tostem cell function were also examined, such as pum1, pum2, nucleostemin,and mili. Orthologous genes have been identified in Drosophila as beingcentral to the maintenance of germline stem cell function in the ovary,such as the RNA binding proteins encoded by piwi and pumilio (Lin, H.(1997) Amu. Rev. Genet. 31: 455-491; Spradling, A. H. et al, (2001)Nature 414: 98-104; Lin, H. (2002) Nature Rev. Genet. 3: 931-940). In C.elegans, loss of function of either piwi orthologs (prg-1 and prg-2) orpumilio orthologs (fbf-1 and fbf-2) depletes germline stem cell, andmammalian orthologs of piwi (miwi/hiwi and mili) and pumilio (pumilio-1and pumilio-2) are known to exist (Cox, D. N. et al. (1998) Genes Dev.12: 3715-3727; Crittenden, S. L. et al. (2002) Nature 417: 660-663;Kuramochi-Miyagawa, S. et al. (2001) Mech. Dev. 108: 121-133; Spassov,D. S. & Jurecic, R. (2002) Gene 299: 195-204).

For ovaries collected at each time point and for control tissues, totalRNA was extracted and 1 μg of total RNA was reverse-transcribed(Superscript II RT; Invitrogen) using oligo-dT primers. Amplificationvia 28 cycles of PCR was performed using Taq polymerase and buffer D(Epicentre) with primer sets specific for each gene (see Table 3). Theribosomal gene L7 was co-amplified and used as a loading control foreach sample, and 28 cycles were found to be within the linear range ofamplification for each experimental primer set.

TABLE 3 Details relating to RT-PCR analyses of gene expression RegionProduct Region Gene Accession # Size Primer Sequence (5′-3′) AmplifiedDmc1 D64107 973 F: gaaggaggatcaagttgtgc    3-976 Dmc1-d3 858 (−d)R: gcttcattttcaggcatctc L7 NM_011291 199 F: ggagctcatctatgagaaggc  209-408 R: aagacgaaggagctgcagaac Scp3 NM_011517 436F: gagccgctgagcaaacatcta   36-472 R: atatccagttcccactgctgc Spo11a, bXM_123992; 431(a) F: ccgaggcctcgttcttcgac   22-453 AF163054 321(b)R: tgtccaccgcagcctggttc Mili AB032605 441 F: tggtactcgagggtggtg2304-2745 R: cagggctcagatttgcag Nucleo- AY181025 600F: cacaagaagcctaggaaggac   120-720 stemin R: ctccaagaagcttccaaaggg Pum1NM_030722 497 F: gcagtgctttggcaggactct   30-527 R: ggcactgtctcgccattgatcPum2 NM_030723 400 F: ggagagagactgcatggggaa  133-533R: gcgacttccaggagtgcgatt

All PCR products were isolated, subcloned and sequenced forconfirmation. In those samples showing more than one amplified productper primer set, each band was isolated, subcloned, and sequenced. Theseadditional bands were determined to be known splice variants of thetargeted genes (i.e., Dmc-1/Dmc-1d; Spo11a/Spo11b).

Past work has demonstrated that expression of SCP3 and DMC1 in germcells is restricted to the zygotene or pachytene stages of meiosis.These stages are earlier than the late diplotene stage, where the firstmeiotic arrest in oocytes is observed. Accordingly, the presence ofpre-diplotene mRNA transcripts, like SCP3 protein, reflects expressionwithin cells other than the oocytes presently arrested in meiosis.Expression levels of SCP3, SPO11 and DMC1 ranged from 6% (SPO11 andDMC1) to 25% (SCP3) of those observed in adult testes (FIG. 3 f), whichis significant considering that daily postnatal germ cell output in thetestis far exceeds that estimated for ovaries. Ovarian expression of allthree meiosis-related genes declined with age (FIG. 3 e), and minimal tono expression of these genes was observed in non-gonadal tissues (FIG. 3g).

Analysis of ovaries collected from mice at various times duringneonatal, juvenile and adult life revealed expression of mili, pumilio-1and pumilio-2, with mai showing an age-related decline in its levels ofexpression (FIG. 4). In addition, expression of nucleostemin, a generecently implicated in stem cell renewal in mammals (Tsai, R. Y. L. andMcKay, R. D. G. (2002) Genes Dev. 16: 2991-3003), was also identified inthe mouse ovary during neonatal, juvenile and adult life (FIG. 4).

Example 3 Post-Natal Ovarian Follicle Renewal

The importance of proliferative germ cells to replenishment of thepostnatal follicle pool was further verified by the use of busulfan, agerm-cell toxicant widely used in spermatogonial stem cellcharacterization in male mice. In the testis, busulfan specificallytargets germline stem cells and spermatogonia, but not post-meiotic germcells, leading to spermatogenic failure. Female rodents exposed in uteroshow a similar gametogenic failure in response to busulfan only if thechemical is given during the window of fetal ovarian germ cellproliferation, as females exposed to busulfan in utero after germ cellproliferation has ceased are born with ovaries that are histologicallyand functionally similar to ovaries of vehicle-exposed mice.

Female mice were injected with vehicle (DMSO) or busulfan (20 mg/kg bodyweight; resuspended in DMSO) on day 25 and again on day 35 postpartum,and ovaries were collected 10 days after the second injection to analyzechanges in non-atretic primordial follicle numbers. Ovaries of femalestreated with busulfan possessed less than 5% of the primordial folliclepool present in vehicle-treated controls 20 days after the start of theexperiment (FIG. 5 a). However, busulfan-exposed ovaries retained anotherwise normal histological appearance, including the presence ofhealthy maturing follicles with non-degenerative oocytes, as well ascorpora lutea, indicative of ovulation (FIGS. 5 b-5 e).

To clarify whether loss of primordial follicles observed inbusulfan-treated females (FIG. 5 a) results from toxicity to existingoocytes, ovaries were collected from female mice at multiple pointsduring and after the busulfan dosing regimen described above, and theywere analyzed for the incidence of primordial follicle atresia. Busulfancaused a slight, transient increase in the number of atretic primordialfollicles, with a plateau of only 46 per ovary 5 days after the firstinjection that quickly declined to basal levels thereafter (FIG. 5 a,inset). This relatively minor and acute atretic response to busulfan wasnegligible considering that over 2,000 primordial follicles were absentin busulfan-exposed ovaries compared with vehicle-treated controls (FIG.5 a). These data reinforce the idea that proliferative germ cells notonly persist in the postnatal ovary, but are also required to routinelyrenew the follicle pool.

To determine the rate of primordial follicle renewal in the postnatalmouse ovary, these results were evaluated in the context of a pastinvestigation of the kinetics of follicle maturation in female mice.Previous analyses demonstrated that the primordial follicle pool isdecreased on average by 89 follicles per day, owing to eitherdegeneration or growth activation to the primary stage of development,between days 14 and 42 postpartum (Faddy, M. J. et al, Cell TissueKinet. (1987) 20: 551-560). In this a comparable window of timedescribed herein (day 16-40 postpartum; see FIG. 1 a), this rate of exitwould be expected to reduce the primordial follicle population by 2,136follicles over this 24-day period. However, the number of primordialfollicles declined by only 294 between days 16 and 40 postpartum (FIG. 1a). The difference between these two values, or 1,842 primordialfollicles, represents the rate of primordial follicle renewal over this24-day period, yielding an average of 77 new primordial follicles perovary per day. Given this calculation, the rate of primordial follicledepletion per day should be the difference between the rate of exit perday provided by previous analyses (89 follicles) and the rate of renewalper day (77 follicles), for a net loss of 12 primordial follicles perovary per day. Using this value, the primordial follicle pool would beexpected to decline between days 16 and 40 postpartum by a total of 288follicles, a number very close to that derived from comparing the actualcounts of non-atretic primordial follicles on day 16 versus day 40(2,334 versus 2,040, or a net loss of 294 primordial follicles; FIG. 1a).

Thus, taken together, busulfan treatment causes a 95% reduction in theresting (primordial) oocyte pool in female mice within three weeks, andthis effect is not due to either enhanced atresia of the primordialoocyte pool.

Although there was no precedence in the literature for busulfan inducingthe growth activation of primordial follicles, and no morphologicalevidence for such an outcome was observed, the number of(growth-activated) primary follicles during the time course has beendetermined and the average ratio of primordial follicles to primaryfollicles over the time course has been calculated. No significantchange in the ratio of primordial to primary follicles was seen betweenbusulfan and vehicle treatment (FIG. 6), indicating that busulfantreatment did not decrease the primordial pool by increasing folliclegrowth activation. These data further support the conclusion thatbusulfan specifically depletes germline stem cell support of oocyteproduction in the ovaries resulting in a gradual loss of the primordialfollicle pool through an absence of oocyte and follicle renewal.

The long-term outcome of anti-cancer treatment on ovarian function inhuman females was also studied. Chemotherapy regimens containingbusulfan result in a near-total incidence of premature ovarian failure(POF), regardless of other drugs used in combination therapy. Forexample, clinical data combined from three studies showed that 20 of 21adolescent girls (mean age=11.5) treated with chemotherapeutic regimenscontaining busulfan experienced hypogonadism indicative of POF, whilecomparable treatments that lacked busulfan caused POF in only 22 of 37girls (mean age=8.7) (Thibaud, E. et al, (1998) Bone Marrow Transplant.21: 287-290; Teinturier, C. et al, (1998) Bone Marrow Transplant. 22:989-994; Afify, Z. et al, (2000) Bone Marrow Transplant 25: 1087-1092).Similarly, 4 of 4 pubertal girls (mean age=13) treated with busulfan incombination therapy showed ovarian damage requiring hormonal replacement(Legault, L. and Bonny, Y. (1999) Pediatric Transplant 3: 60-66). In yetanother study, busulfan treatment in women between the ages of 16 and 40(median age=30) caused POF in 19 of 19 cases (Sanders J. E. et al,(1996) Blood 87 3045-3052). Moreover, in a study where combined busulfanand cyclophosphamide therapy was compared to cyclophosphamide alone, 72or 73 patients treated with both agents exhibited POF (ages 14-57,median=38) while cyclophosphamide alone resulted in POF in 47 of 103patients (ages 13-58, median=28) (Grigg, A. P. et al, (2000) Bone MarrowTransplant 26: 1089-1095).

Exposure to busulfan resulted in POF in 115 of 117 patients, whilecomparable chemotherapy treatments lacking busulfan were associated withPOF in only 69 of 140 cases. While busulfan treatment may cause POF inhumans by accelerating oocyte loss (death), based on the findings withbusulfan in female mice, the results from these clinical trials withbusulfan may also indicate an irreversible destruction of human femalegermline stem cells leading to POF.

The existence of mammalian female germline stem cells implies aninherent capacity of the ovaries to generate, or regenerate following aninsult, new stockpiles of resting (primordial) oocyte-containingfollicles in a regulated fashion. To address this issue directly, adultfemale mice were injected with doxorubicin to synchronize oocyte death(Perez, G. I. et al, (1997) Nature Med. 3, 1228-32), and ovaries werecollected at multiple intervals after drug exposure to assess germ celldynamics. As expected, a rapid and extensive loss of primordial andearly growing follicles (oocytes) occurred within the first 24 h afterdoxorubicin treatment (FIG. 7 a,b). However, a spontaneous regenerationof both the primordial and total immature follicle pools was observedbetween 24 and 36 h post-treatment, and the number of oocyte-containingimmature follicles stabilized thereafter (FIG. 7 a,b).

To further show that the adult mammalian ovary is fully capable ofde-novo oocyte production, a recent report showing that inhibition ofhistone deacetylation rapidly expands haematopoietic stem cells (Milhem,M. et al. (2004) Blood 103, 4102-4110) was used as a basis to testwhether acute in-vivo suppression of histone deacetylase (HDAC) activitycould similarly enhance germline stem cell function. Prepubertal femalemice were given a single intraperitoneal injection of the broad-spectrumhistone deacetylase inhibitor, Trichostatin A (TSA; 10 mg/kg bodyweight, resuspended in DMSO), or vehicle (DMSO). Animals were sacrificed24 hours post-injection, histological preparations of ovaries wereprepared, and oocyte-containing follicles were counted per standardlaboratory procedures (see Example 1). Treatment with TSA caused a 53%increase in the number of total healthy immature oocyte-containingfollicles per ovary when compared with ovaries of control mice given thevehicle treatment. Not only was the resting (primordial) pool offollicles increased by 42%, but also the number of early growing(primary) follicles was increased (FIG. 7 c).

Treatment with TSA either reduced the incidence of mature follicle loss(death or atresia) or increased new oocyte and follicle production bygermline stem cells. Since the average baseline level of immaturefollicle atresia in untreated 13-day old mice is 16±4 (n=4), a decreasein the rate of atresia cannot explain the large increase (more than1,600) in healthy immature follicles. Without new oocyte production, anincrease in the number of primordial oocyte-containing follicles isimpossible. Notably, should the production of new oocytes not occur, theincrease in primary follicles must be subtracted from their source, i.e.the number of primordial follicles. As the number of primordialfollicles does not decrease but instead increases, the only explanationfor the dramatic increase in oocyte and follicle numbers following TSAexposure is a significant new production of immature oocytes fromgermline stem cells.

More striking results were obtained in adults; in that TSA increasedprimordial follicle numbers in female mice at 240 days of age by 89%within 24 h (FIG. 7 d). Since the observed increases could not beattributed to either a reduced rate of primordial follicle growthactivation to the primary stage of development (FIG. 7 c,d) or a reducedincidence of atresia (FIG. 7 e), these data provide additional evidencethat oogenesis and folliculogenesis persist during adult life inmammalian females.

Example 4 Evidence of Post-Natal Ovarian Folliculogenesis

Transgenic mice with ubiquitous expression of GFP (obtained from Jacksonlaboratories, strain STOCK TgN(GFPU)5Nagy) were used to provideadditional evidence for ongoing folliculogenesis in postnatal life.Heterozygous transgenic male and female mice with ubiquitous expressionof GFP were mated to generate wild-type and transgenic female offspringfor intrabursal ovarian grafting. Briefly, young adult (58-69 dayspostpartum) transgenic female mice were anesthetized (avertin, 200 mgper kg, intraperitoneal) to expose one of the two ovaries in each mousethrough dorso-lateral incisions. For each animal, a small hole was cutin the ovarian bursa laterally near the hilus, and approximatelyone-half of the host ovary was removed in preparation for grafting.Ovaries collected from donor (wild-type littermate) female mice werebisected, and one-half of a wild-type ovary was placed within thetransgenic recipient's bursal cavity in contact with the remaining hostovarian tissue. The reproductive tract was then allowed to settle backinto the peritoneal cavity and the incision was closed. A total of sixtransgenic hosts were used for this experiment, four of which receivedunilateral wild-type ovarian grafts while the remaining two receivedbilateral wild-type ovarian grafts. Between 3-4 weeks after surgery, theovarian tissues were removed and processed for GFP visualization, afterpropidium iodide counterstaining, by confocal laser scanning microscopy.The grafted ovarian fragments, upon gross visual inspection, showedevidence of neovascularization and adhesion to the host ovarian tissue(FIG. 8). Confocal microscopic analysis revealed follicle-enclosed,GFP-positive oocytes in the wild-type ovarian fragments that wereindistinguishable from follicle-enclosed oocytes in the host ovariantissue (FIGS. 9 and 10). Moreover, the granulosa cells enveloping theGFP-positive oocytes in the grafts were negative for GFP, indicatingthat transgenic germ cells had infiltrated the grafted tissue andinitiated folliculogenesis with the resident wild-type somatic cells.

To sustain the addition of new primordial follicles during juvenile andadult life, the mouse ovary must possess either a small pool ofasymmetrically dividing germline stem cells or a large pool ofnon-renewing, pre-meiotic germ cells that produce oocytes aftersymmetric divisions. Histomorphometric studies at day 30 postpartumrevealed the presence of 63±8 such cells per ovary (mean±standard error,n=4 mice), a number close to that expected for a small pool ofasymmetrically dividing germ cells.

Example 5 Oocyte Dynamics in Transgenic Mouse Models

Published data from the analysis of oocyte dynamics in the Bax knockoutmouse (Perez et al., (1999) Nature Genetics 21: 200-203), andunpublished contemporary data from the Caspase-6 knockout mouse wasre-evaluated and compared in view of the results demonstratingpost-natal oocyte folliculogenesis presented herein. Data shown hereprovide additional evidence of germline stem cell function and, for thefirst time, reveal the effects of these apoptosis regulatory geneknockout mice upon germline stem cell production of new oocytes.Histological preparations of ovaries were prepared, andoocyte-containing follicles were counted per standard laboratoryprocedures (Tilly, J. L. (2003) Reprod Biol Endocrinol 1: 11; see alsoExample 1). The Bax protein has been shown to be a crucial pro-apoptoticmolecule within somatic cells in the ovary and in oocytes (Tilly, J. L.(1996) Rev Reprod 1: 162-172; Perez, G. I. et al., (1997) Nature Med. 3:1228-1232). Bax knockout mice were shown to have greatly extendedovarian function, with 200 to 300 non-atretic follicles present in theovaries of 20-22 month-old females compared to essentially zero inage-matched wild-type controls. Data comparing immature folliclesnumbers during early postnatal life (Day 4) and early reproductiveadulthood are shown above. While oocyte endowment in early life iscomparable between Bax-null and wild-type mice, Bax-null mice havenearly 2.5 times greater primordial follicles at Day 42 than wild-type,and a significantly greater number of primary follicles as well (FIG.11). Concurrent measurement of atresia, or death, in these mice at Day42 revealed a major decrease in primordial follicle atresia. Thus, theBax-null mice fail to eliminate the normal number of primordialfollicles in comparison to wild-type mice.

Caspase-6 is also a pro-apoptotic molecule, functioning as a proteasethat cleaves structural intracellular targets during the onset ofapoptosis (Ruchaud, S. et al. (2002) EMBO J 21: 1967-1977). The mouseknockout of Caspase-6 does not result in an overt phenotype. Recently,however, Caspase-6-null mice were shown to demonstrate an ovarianphenotype similar to Bax-null mice, in that Day 4 similar numbers ofimmature follicles are present in both Caspase-6-null ovaries and thoseof wild-type mice (FIG. 12). Caspase-6-null mice also have a significantincrease in the number of primordial and a large increase in the numberof total immature follicles at Day 42 of life. However, when immaturefollicle atresia was measured in these mice at Day 42, unlike theBax-null mouse, Caspase-6-null mice show no change in the amount ofatresia. Since a decrease in the amount of atresia is not seen, the onlyother explanation for the increase in follicle numbers in Caspase-6-nullmice is an increase in the production of new oocytes due to the deletionof Caspase-6. Caspase-6 is therefore a regulator of germline stem cellapoptosis.

As shown herein, Bax is a regulator of oocyte death. Given thatprimordial follicle atresia is only halved in Bax-null mice, and thatBax-null ovaries contain oocytes for as many as 10 to 14 months longerthan wild-type ovaries, Bax also regulates germline stem cell apoptosis.In contrast, Caspase-6 can be directly seen to regulate germline stemcell function/death but does not regulate oocyte death. Thus, Caspase-6and Bax are regulator(s) of oocyte production at the level ofoocyte-progenitor germline stem cells. Modulating germline stem cellfunction by modulating the function of key apoptotic regulators in vivois thus an important strategy for the extension of ovarian function inmammals.

To further demonstrate the existence of postnatal female germline stemcells, the ovaries of wild-type mice were compared with ovaries fromAtaxia-telangiectasia mutation (Atm) gene-deficient mice. Atm-deficientmale and female mice have been shown to be infertile due to the completeloss of the production of mature gametes, e.g. sperm and oocytes(Barlow, C. et al. (1996) Cell 86: 159-171). Atm-deficiency was shown toresult in aberrant early stages of meiosis, detected as early as theleptotene stage, that results in increased apoptosis of developinggametes (Barlow, C. et al. (1998) Development 125: 4007-17) andtherefore total gamete loss. Ovaries from Atm-deficient females wereshown to be completely barren of oocytes and follicles by 11 days of age(Barlow, C. et al. (1998) Development 125: 4007-17). Representativehistology of postpartum Day 4 wild-type (FIG. 13A, magnified in C) andAtm-null (FIG. 13B, D) ovaries is depicted in FIG. 13. Thus, if alloocyte production has occurred prenatally and has resulted in a fixedpool of diplotene oocytes within primordial follicles at birth, andAtm-deficiency results in a complete lack of oocytes, no germline oroocyte marker gene expression should occur in these “barren” ovaries.However, due to detection of germline stem cells in the postnatal femaleovary, pre-meiotic germline stem cells can be present and capable ofself-renewal, but ongoing oocyte production is impossible due to meioticentry in the absence of Atm. The expression of germline markers in theAtm-deficient ovary versus wild-type controls was performed byreverse-transcription followed by PCR and representative data (n=3) isshown in FIG. 13, right panel. As predicted, the pluripotency markerOct-4 (Brehm, A. et al. (1998) APMIS 106: 114-126) and the germlinemarkers Dazl (McNeilly, J. R. et al. (2000) Endocrinology 141:4284-4294; Nishi, S. et al. (1999) Mol Hum Reprod 5: 495-497) and Stella(Bortvin, A. et al. (2004) BMC Dev Biol 23: 2) are all expressed in theAtm-deficient ovary at postnatal Day 71. Semi-quantitative comparison ofthe relative levels of these genes by examination of the loading controlL7 shows that, as expected, these genes are expressed at much lowerlevels than in wild-type ovaries containing oocytes. The contralateralovary in each animal used for RT-PCR analysis was prepared forhistology, and the sampling and examination of histological sectionsfrom Atm-null mice did not reveal any oocytes or structures resemblingfollicles as expected. Thus, Atm-deficiency results in a pool ofgermline stem cells that may renew until at least several weeks of adultlife (Day 71) but may not, as reported, produce viable oocytes due tothe meiotic defect that results in gamete death.

Example 6 Isolation and Characterization of Cells from Adult MouseOvaries

Stage-Specific Embryonic Antigen-1 (SSEA-1) has been shown to decoratethe surface of specialized mammalian cells, notably embryonic stem cells(Henderson, J. K. et al., (2002) Stem Cells 20: 329-37; Furusawa, T. etal., (2004) Biol Reprod. 70: 1452-7) and primordial germ cells (“PGC”)(Matsui, Y. et al., (1992) Cell 70: 841-7; Gomperts, M. et al., (1994)Development. 120: 135-41). Thus, SSEA-1 was a potential marker forfemale germline stem cells and their progenitors. Furthermore, fetal PGCexpression of SSEA-1 correlates with the well-established developmentalperiod in which germ cells (female and male) are pre-meiotic and able todivide. By extension, postnatal female germline stem cells, alsopre-meiotic and able to divide, could also express SSEA-1.

Immunohistochemical detection of SSEA-1 in the mouse ovary (adult andprepubertal) revealed a small central population of SSEA-1 positivecells in the core or medullary region of the ovary (FIG. 14). However,outside of a low level of immunoreactivity occasionally observed in somescattered granulosa cells, SSEA1 was not expressed in any other area ofthe ovary or in any cell type of known origin, including oocytes. Thesecells are otherwise unremarkable when compared morphologically toneighboring cells, which share their stromal appearance; SSEA-1immunoreactivity now reveals their stem cell properties. SSEA-1 wastherefore selected as a marker to be used in the isolation of thispopulation of cells expected to be postnatal female germline stem cellsor the progenitors thereof.

A strategy for the isolation is schematically shown in FIG. 15. Adultovaries (postnatal day 51) were removed and homogenized as follows. Eachovary was placed in 250 μl DMEM media (Gibco #11995-06), pre-warmed to37° C., and was torn apart with a syringe needle and forceps. Care wastaken to leave macroscopic growing follicles intact, and maximaldisruption of medullary/stromal structures was attempted.Two-hundred-and-fifty μl of pre-warmed 2×-concentrated homogenizationmedium (DMEM+1 mg/ml collagenase [Gibco #17100-017]) was added to thedish containing the disrupted ovary, and tissue/media were transferredto a 15 ml conical tube. Tissues in homogenization media were incubatedwith shaking for 45 minutes at 37° C. The digested tissue and cells werespun through a 40-micron cell strainer for 10 minutes at 1000×g. Mediumwas removed, and the pelleted cells from each homogenized ovary werere-suspended in 500 μl phosphate buffered saline (“PBS”)-0.1% bovineserum albumin (“BSA”) and cooled to 4° C. prior to subsequentimmunomagnetic separation.

The cells were isolated from the ovarian homogenate using anti-mouse IgMbeads (Dynabeads M-450 Rat anti-mouse IgM; Dynal Biotech), pre-coated byadding 3 μg of anti-SSEA-1 antibody per 50 μl aliquot of beads andincubating for 15 minutes at 4° C. with rocking. Coated beads werewashed 3 times with PBS-0.1% BSA and pelleted using the Dynal MPC, andthen resuspended in PBS-0.1% BSA. Afterwards, 12.5 μl of coated beadswere then added to each 500 μl aliquot of ovarian cells. Cells and beadswere incubated with gentle rocking at 4° C. for 30 minutes. Cells boundto the beads were isolated by washing 3 times in PBS-0.1% BSA afterpelleting with the Dynal MPC. After the last separation, the supernatantwas removed and the beads, including bound cells, were resuspended in250 μl Tri Reagent (Sigma, T9424), vortexed, and stored at −80° C. priorto RNA isolation.

The SSEA-1 positive, isolated cellular fraction was used forreverse-transcription of messenger RNA/polymerase chain reactionamplification (RT-PCR) to determine their gene expression profile. TotalRNA was extracted from each sample and 1 μg was reverse transcribed(Superscript II RT; Invitrogen) using oligo-dT primers. Amplificationvia 28-35 cycles of PCR was then performed using Taq polymerase andBuffer-D (Epicentre) with primer sets specific for each gene (Table 4a,b). For each sample, RNA encoded by the ribosomal gene L7 was amplifiedand used as a loading control (‘house-keeping’ gene). All PCR productswere isolated, subcloned and sequenced for confirmation.

TABLE 4a RT-PCR analysis of gene expression in mouse tissues ProductRegion Gene Accession # Size Primer Sequence (5′-3′)¹ Amplified² DazlNM_010021 317 F: gtgtgtcgaagggctatggat 230-547 R: acaggcagctgatatccagtgFragilis NM_025378 150 F: gttatcaccattgttagtgtcatc 355-505F: aatgagtgttacacctgcgtg Gdf9 L06444 708 F: tgcctccttccctcatcttg747-1454 R: cacttcccccgctcacacag Hdac6 NM_010413 383F: acgctgactacattgctgct 944-1327 R: tctcaactgatctctccagg L7 NM_011291199 F: ggagctcatctatgagaaggc 209-408 R: aagacgaaggagctgcagaac MvhNM_010029 212 F: ggaaaccagcagcaagtgat 479-691 R: tggagtcctcatcctctggOct4 X52437 589 F: cccaagttggcgtggagactt 158-747R: cttctggcgccggttacagaa Scp3 NM_011517 436 F: gagccgctgagcaaacatcta 36-472 R: atatccagttcccactgctgc Stella AY082485 353F: cccaatgaaggaccctgaaac  27-380 R: aatggctcactgtcccgttca Zp3 M20026 182F: ccgagctgtgcaattcccaga  50-232 R: aaccctctgagccaagggtga

The SSEA-1 isolated fraction shown in FIG. 16 is a fraction of cellsthat express genes denoting pluripotency (Oct-4: Brehm, A. et al.,(1998) APMIS 106: 114-126), and placing their lineage within thegermline (Dazl: McNeilly, J. R. et al., (2000) Endocrinology 141:4284-4294, Nishi, S. et al., (1999) Mol Hum Reprod 5: 495-497; Stella:Bortvin, A. et al., (2004) BMC Dev Biol 23: 2; and the mouse Vasahomologue, Mvh: Fujiwara, Y. et al. (1994) Proc. Natl. Acad. Sci. USA91, 12258-12262). This fraction does not express genes found in eithergrowing oocytes (e.g., GDF-9: Dong, J. et al., (1996) Nature 383:531-535; and ZP3: Dean, J. (2002) J. Reprod. Immunol. 51(1-2) 171-80) orin resting primordial oocytes (e.g., HDAC6) (FIG. 16). All of thesegenes are, as expected, expressed in the SSEA-1 depleted fraction ofcells as this fraction contains oocytes. Moreover, since the SSEA-1isolated fraction of cells does not express genes found in eitherresting primordial oocytes or growing oocytes, this fraction is notcontaminated with oocytes. In addition, these cells also do not expressthe synaptonemal complex protein SCP3, a marker of meiotic entrance(Yuan, L. et al., 2000 Mol Cell 5: 73-83; Johnson, J. et al., 2004Nature 428: 145-150) supporting their identification as female germlinestem cells and/or their progenitors.

Separately, live female germline stem cells and/or their progenitorswere isolated from previously described ovarian homogenates (see above)using the above methodology with slight modifications. In this case, theanti-SSEA-1 antibody was biotinylated through the long-chainN-hydroxysuccinimide ester of biotin with primary amine reactivity(NHS-LC-biotin). For a full overview of biotinylation procedures, seethe Pierce Catalogue and Handbook (Pierce, Rockford, USA).

The CELLection biotin binder kit from Dynal Biotech was then used toisolate the SSEA-1 positive cells. The CELLection beads are preferablewhere post-isolation removal of affinity beads from the cells isdesired. The methods are briefly described as follows. CELLection beadswere re-suspended thoroughly and 50 μl aliquots were transferred to atube suitable for the Dynal MPC. The tube was placed in the Dynal MPCfor 1 minute, removed and 1-2 ml buffer (e.g., PBS with 0.1% Tween-20)was added for re-suspension.

To coat the beads with biotinylated anti-SSEA-1 antibody, 2-3 μg ofbiotinylated antibody and 50 μl of beads were combined in a tube androtated for 30 minutes at room temperature. The tube was then placed inthe Dynal MPC for 1 minute to pellet the beads coated with anti-SSEA-1antibody. The beads were washed with 1 ml PBS with 0.1% Tween-20, andpelleted using the Dynal MPC, three times. Coated beads werere-suspended in the original volume of PBS with 0.1% BSA), giving afinal concentration of 4×10⁸ beads/ml (note that 0.02% sodium azide canbe optionally added as a preservative).

As detailed above, cells from one ovary were re-suspended in 500 μlPBS-0.1% BSA and cooled to 4° C., after which 12.5 μl of anti-SSEA-1coated beads was combined with each 0.5 ml ovary homogenate sample.Cells and beads were incubated with gentle rocking at 4° C. for 30minutes. Cells bound to the beads were isolated by washing 3 times inPBS-0.1% BSA after pelleting with the Dynal MPC. The tube was removedfrom the Dynal MPC and rosetted cells were combined with RPMI 1640(containing 1% FCS). Rosetted cells were re-suspended by pipetting,transferred to a new vial and placed in the Dynal MPC for 1 minute. Thetube was removed from the Dynal MPC and cells were resuspended bypipetting the rosetted cells in a minimum of 500 μl RPMI (containing 1%FCS). This step was repeated twice. After the final wash, the fluid wasremoved and the rosetted cells were re-suspended in RPMI (1% FCS)pre-warmed to 37° C. Releasing buffer was added at 2 μl, and the mixturewas incubated for 15 minutes at room temperature with gentle tilting androtation. Rosettes were flushed vigorously through a pipette 8 times,and then placed in the Dynal MPC for 1 minute. Supernatant containingthe released cells was pipetted to a new test tube containing 200 μlRPMI (with 10% FCS). Aliquots of cells (e.g., 50-100 μl) were collectedand stored for future use for in vitro culture and transplantation torecipient animals.

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1-13. (canceled)
 14. A method of in vitro fertilization of a femalesubject, said method comprising the steps of: a) producing an oocyte byculturing an isolated cell that is mitotically competent and expressesVasa, Oct-4, Dazl, Stella and optionally, a stage-specific embryonicantigen in the presence of an agent that differentiates the cell into anoocyte; b) fertilizing the oocyte in vitro to form a zygote; and c)implanting the zygote into the uterus of a female subject.
 15. A methodof oocyte production, comprising culturing the cell of claim 14 in thepresence of an agent that differentiates the cell into an oocyte,thereby producing an oocyte.
 16. (canceled)
 17. A pharmaceuticalcomposition comprising a purified population of cells of claim 14 and apharmaceutically acceptable carrier. 18-21. (canceled)
 22. A method ofoocyte production in a subject, comprising providing the pharmaceuticalcomposition of claim 17 to a tissue of the subject, wherein the cellsengraft into the tissue and differentiate into oocytes, therebyproducing oocytes in the subject.
 23. (canceled)
 24. A method ofinducing folliculogenesis in a subject, comprising providing thepharmaceutical composition of claim 17 to the subject, wherein the cellsengraft into a tissue of the subject and differentiate into oocyteswithin follicles, thereby inducing folliculogenesis in the subject. 25.(canceled)
 26. A method of treating infertility in a female subject inneed thereof comprising administering a therapeutically effective amountof the pharmaceutical composition of claim 17 to the subject, whereinthe cells engraft into the ovary and differentiate into oocytes, therebytreating infertility.
 27. A method of repairing damaged ovarian tissue,comprising providing a therapeutically effective amount of thepharmaceutical composition of claim 17 to the tissue, wherein the cellsengraft into the tissue and differentiate into oocytes, therebyrepairing the damaged tissue. 28-30. (canceled)
 31. A method ofrestoring ovarian function in a menopausal female subject, comprisingadministering a therapeutically effective amount of the pharmaceuticalcomposition of claim 17 to the subject, wherein the cells engraft intothe ovary and differentiate into oocytes, thereby restoring ovarianfunction in the subject.
 32. (canceled)
 33. A method for oocyteproduction in a subject, comprising contacting female germline stemcells, or their progenitor cells, of the subject with an agent thatdifferentiates the female germline stem cells, or their progenitorcells, into oocytes, thereby producing oocytes in the subject. 34.(canceled)
 35. (canceled) 36-39. (canceled)
 40. A method for oocyteproduction in a subject, comprising contacting ovarian tissue of thesubject with an agent that increases the amount of female germline stemcells, or their progenitor cells, thereby producing oocytes in thesubject.
 41. (canceled)
 42. (canceled)
 43. A method of treatinginfertility in a female subject in need thereof comprising contactingovarian tissue of the subject with an agent that increases thedifferentiation of female germline stem cells, or progenitor cellsderived from female germline stem cells, into oocytes, thereby treatinginfertility in the subject.
 44. A method of treating infertility in afemale subject in need thereof comprising contacting ovarian tissue ofthe subject with the agent of claim 40, thereby treating infertility inthe subject.
 45. (canceled)
 46. A method of restoring ovarian functionin a post-menopausal female subject comprising contacting ovarian tissueof the subject with the agent of claim 40, thereby restoring ovarianfunction in the subject.
 47. (canceled)
 48. A method of reducing theamount of female germline stem cells, or their progenitor cells, in asubject comprising contacting female germline stem cells, or theirprogenitor cells, in the subject with an agent that reduces cellproliferation, thereby reducing the amount of female germline stemcells, or their progenitor cells, in the subject.
 49. (canceled)
 50. Amethod of reducing the amount of female germline stem cells, or theirprogenitor cells, in a subject comprising contacting female germlinestem cells, or their progenitor cells, in the subject with an agent thatinhibits cell survival, thereby reducing the amount of female germlinestem cells, or their progenitor cells, in the subject. 51-54. (canceled)55. A method of reducing the amount of female germline stem cells, ortheir progenitor cells, in a subject comprising contacting femalegermline stem cells, or their progenitor cells, in the subject with anagent that promotes cell death, thereby reducing the amount of femalegermline stem cells, or their progenitor cells, in the subject. 56-61.(canceled)
 62. A method of providing contraception to a female subjectcomprising contacting ovarian tissue of the subject with an agent thatdecreases the amount of female germline stem cells, or their progenitorscells, thereby providing contraception to the subject.
 63. (canceled)64. A method of isolating of a composition comprising female germlinestem cells and their progenitor cells, said method comprising the stepsof: a) homogenizing ovarian tissue; b) contacting the tissue with anagent that binds to a stage-specific embryonic antigen; and c) isolatingfemale germline stem cells and their progenitor cells.
 65. (canceled)